Chlamydia pneumoniae polynucleotides and uses thereof

ABSTRACT

The subject of the invention is the genomic sequence and the nucleotide sequences encoding polypeptides of  Chlamydia pneumoniae,  such as cellular envelope polypeptides, which are secreted or specific, or which are involved in metabolism, in the replication process or in virulence, polypeptides encoded by such sequences, as well as vectors including the said sequences and cells or animals transformed with these vectors. The invention also relates to transcriptional gene products of the  Chlamydia pneumoniae  genome, such as, for example, antisense and ribozyme molecules, which can be used to control growth of the microorganism. The invention also relates to methods of detecting these nucleic acids or polypeptides and kits for diagnosing  Chlamydia pneumoniae  infection. The invention also relates to a method of selecting compounds capable of modulating bacterial infection and a method for the biosynthesis or biodegradation of molecules of interest using the said nucleotide sequences or the said polypeptides. The invention finally comprises, pharmaceutical, in particular vaccine, compositions for the prevention and/or treatment of bacterial, in particular  Chlamydia pneumoniae,  infections.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Provisional Application Ser. No. 60/107,078, filed Nov. 4, 1998.

The Sequence Listing for this application is on duplicate compact discs labeled “Copy 1” and “Copy 2.” Copy 1 and 2 each contain only one file named “seqlist-28July2001.txt” which was created on Jul. 30, 2001, and is 5,284 KB. The entire contents of each of the computer discs are incorporated herein by reference in their entireties.

The subject of the invention is the genomic sequence and the nucleotide sequences encoding polypeptides of Chlamydia pneumoniae, such as cellular envelope polypeptides, which are secreted or specific, or which are involved in metabolism, in the replication process or in virulence, polypeptides encoded by such sequences, as well as vectors including the said sequences and cells or animals transformed with these vectors. The invention also relates to transcriptional gene products of the Chlamydia pneumoniae genome, such as, for example, antisense and ribozyme molecules, which can be used to control growth of the microorganism. The invention also relates to methods of detecting these nucleic acids or polypeptides and kits for diagnosing Chlamydia pneumoniae infection. The invention also relates to a method of selecting compounds capable of modulating bacterial infection and a method for the biosynthesis or biodegradation of molecules of interest using the said nucleotide sequences or the said polypeptides. The invention finally comprises, pharmaceutical, in particular vaccine, compositions for the prevention and/or treatment of bacterial, in particular Chlamydia pneumoniae, infections.

Comparative analysis of the sequence of the gene encoding the ribosomal 16S RNA has been widely used for the phylogenetic study of prokaryotes. This approach has made it possible to classify the Chlamydiae among the eubacteria, among which they represent a well-isolated group, with, nevertheless, a very weak link with the planctomyces. The Chlamydiae thus exhibit some unique characteristics within the eubacteria, in particular their development cycle and the structure of their membranes. They have a unique two-phase cell cycle: the elementary body, a small extracellular form, attaches to the host and is phagocytosed; in the phagosome, it is converted to the replicative intracellular form, the reticulate body. The Chlamydiae are obligate intracellular bacteria which multiply in eukaryotic cells at the expense of their energy reserves and nucleotide pools; they are responsible for a wide variety of diseases in mammals and birds. The Chlamydiae are the only members of the order Chlamydiales, of the family Chlamydiaceae and of the genus Chlamydia. Within the genus Chlamydia, four species are currently described: Chlamydia trachomatis, Chlamydia psittaci, Chlamydia pneumoniae and Chlamydia pecorum. These bacteria are grouped together and share biological and biochemical properties. Among them, only the first three infect humans, Chlamydia pecorum being a pathogen of ruminants.

The species Chlamydia psittaci infects many animals, in particular birds, and is transmissible to humans. It is responsible for atypical pneumonia, for hepatic and renal dysfunction, for endocarditis and for conjunctivitis.

The species Chlamydia trachomatis is the best characterized. Besides a murine strain, it is divided into two groups which are distinguishable by the nature of the diseases for which they are responsible: trachoma, genital attack and venereal lymphogranulomatosis. There are fifteen human serotypes of Chlamydia trachomatis (A, K) and LGV (L1, L2, L3). Strains A to C are mainly found in eye infections, whereas strains D to K and LGV are essentially responsible for genital entry infections. It should be mentioned that the LGV strains are responsible for systemic diseases. Historically, it was in 1906 that Halberstaeder and on Provaseck discovered, in trachoma patients, the presence of inclusions in the cytoplasm of the cells derived from conjunctival scrapings. In 1940, Rake and Jones described these same inclusions in certain cells obtained by puncturing the ganglia from a patient suffering from venereal granulomatosis. Characterization of the Chlamydia trachomatis microorganism was only successfully carried out in 1957, after a series of isolations in cell cultures.

It was in 1983 that Chlamydia pneumoniae was recognized as a human pathogen (Grayston J T et al., 1986); since then, special attention has been paid to this bacterium and it is estimated (Gaydos C A et al., 1994) that 10% of pneumonias, and 5% of bronchitides and sinusites are attributable to Chlamydia pneumoniae (Aldous M B et al., 1992). More recently, the association of this bacterium with the pathogenesis of asthmatic disease and of cardiovascular impairments is increasingly of interest.

Serological studies have made it possible to observe that Chlamydia pneumoniae infection is common in children between 5 and 16 years of age. Before this age, it is rare to find antibodies; the increase in the number of individuals carrying antibodies is then correlated with age up to 20 years. Accordingly, 50% of adults are carriers of antibodies, it being possible for this prevalence to be as high as 75%. These figures are all the more striking since a first infection induces antibody levels of which the persistence over time is limited to 3 or at most 5 years, which suggests frequent reinfection during the entire lifespan. The annual seroconversion rate is about 8% between 8 and 12 years and about 6% between 12 and 16 years (Haidl et al., 1994). Before the age of 15 years, the seroprevalence of the disease is identical between both sexes. After this age, men are more frequently infected than women; this is true in all regions worldwide where such studies have been carried out.

These infections are geographically highly widespread, as shown by numerous studies carried out throughout the world (Kanamoto Y et al., 1991; Tong C Y et al., 1993). Developed countries of the north such as Canada, Denmark and Norway have the lowest infection rates; conversely, the highest prevalence rates are found in the less developed countries of tropical regions where the infection may occur before the age of 5 years.

Humans are the only known reservoir for Chlamydia pneumoniae and it is probable that the infection is caused by direct transmission, respiratory secretions probably being responsible for this low-yield transmission (Aldous et al., 1992). The chain of transmission may also appear to be indirect (Kleemola M et al., 1988), suggesting that the infection is caused by an effective transmission, but also that asymptomatic carriers exist, which could explain the high prevalence of the disease. Other studies (Mordhorst C H et al., 1992) show that the efficiency of the transmission varies according to the individuals and list cases of infection affecting all or the majority of members of one family or of a group of families. The period of incubation is several weeks, significantly longer in this regard than that of many other respiratory pathogenic agents. Although under conditions of high relative humidity the infectivity of Chlamydia pneumoniae in the open air decreases rapidly, suggesting a direct mode of transmission under these conditions, it is probable that the transmission occurs in some cases indirectly since the microorganism can survive for up to 30 hours in a hostile environment (Falsey et al., 1993).

Clinical manifestations due to Chlamydia pneumoniae are essentially respiratory diseases. Pneumonia and bronchitis are the most frequent because they are clinically patent: since etiological diagnosis is evoked in this case, the infectious agent is identified. The asymptomatic diseases are probably numerous (Grayston J T et al., 1992; Grayston J T et al., 1986; Thom D H et al., 1990). The disease then progresses via bronchitis or pneumonia; fever is absent at the time of examination but is sometimes reported by the patient. The degree of seriousness of the disease is variable and in hospitalized patients, it is common to observe pleural effusion; a generalized infection may also be observed and, in severe cases, anatomicopathological examination shows Chlamydia pneumoniae diseases.

Other syndromes such as sinusitis (Hashiguchi K et al., 1992), purulent otitis media (Ogawa H et al., 1992), or pharyngitis (Huovinen P et al., 1989) have been described, as well as infections with respiratory impairments similar to asthma (Hahn D L et al., 1991). Chlamydia pneumoniae has also been associated with sarcoidosis, with erythema nodosum (Sundelof et al., 1993) and one case of Guillain-Barrésyndrome has even been described (Haidl et al., 1992). The involvement of Chlamydia pneumoniae in Reiter's syndrome has also been evaluated (Braun J et al., 1994).

The association of Chlamydia pneumoniae with coronary diseases and with myocardial infarction was first suspected from the observation of the high antibody level in 71% of patients having a heart disease (Shor A et al., 1992; Kuo C C et al., 1993; Puolakkainen M et al., 1993; Thomas G N et al., 1997). Studies carried out in several countries have shown similar results in patients with atheromatous impairments (Shor A et al., 1992; Kuo C C et al., 1993; Puolakkainen M et al., 1993; Grayston J T et al., 1996; Casas-Ciria J et al., 1996; Thomas G N et al., 1997; Jackson L A et al., 1997) and in patients with carotid impairments. Anatomicopathological and microbiological studies have detected Chlamydia pneumoniae in the vessels. The electron microscope has made it possible to visualize the bacterium (Ladany S et al., 1989), which has in fact been demonstrated by other techniques such as PCR (Campbell L A et al., 1992; Kuo C C et al., 1993; Kuo C C et al., 1988). It also appears that the bacterium is more frequently found in old atheromatous lesions. Other studies carried out on young subjects from 15 to 35 years have given the opportunity to study the coronary arteries of people without atherosclerosis, this observation not being possible in older subjects (the onset of the atheromatous disease is early). In these young subjects, the PCR studies did not find Chlamydia pneumoniae in subjects free of atheromatous disease, but revealed the presence of Chlamydia pneumoniae in two of the eleven subjects who showed early lesions and in six of the seven subjects who developed atheroma plaques. These studies therefore show that the atheroma plaque is very strongly correlated with the presence of Chlamydia pneumoniae, but the role played by the bacterium in vascular pathology is not yet defined.

The data relating to controlled clinical studies analysing the effect of treatments in Chlamydia pneumoniae infections are limited in number. Unlike penicillin, ampicillin or the sulphamides, erythromycin, tetracycline or doxycycline show an antibiotic activity in vitro against Chlamydia pneumoniae. However, a treatment at high doses should be continued for several weeks in order to avoid a recurrence of the infection. Accordingly, the use of two new macrolides, clarithromycin and azithromycin, whose diffusion, bioavailability and half-life allow shorter and better tolerated cures, is nowadays preferred. In the absence of definitive proof based on the results of clinical studies, an effective, without recurrences, and well-tolerated treatment of Chlamydia pneumoniae infections therefore remains desirable.

An even more important need up until now relates to a specific and sensitive diagnosis, which can be carried out conveniently and rapidly, allowing early screening for the infection. Methods based on Chlamydia pneumoniae culture are slow and require a considerable know-how because of the difficulty involved in the collection, preservation and storage of the strain under appropriate conditions. Methods based on antigen detection (EIA, DFA) or on nucleic acid amplification (PCR) provide tests which are more suitable for laboratory practice. A reliable, sensitive and convenient test, which allows distinction between serogroups and a fortiori between Chlamydia pneumoniae species is therefore highly desirable.

This is all the more important since the symptoms of Chlamydia pneumoniae infection appear slowly, since all the pathologies associated with these infections have not yet been identified, and since, as has been mentioned above, an association is suspected between these infections and serious chronic infections, asthma or atherosclerosis.

No vaccine is yet available against Chlamydia pneumoniae: this is due to the labile nature of the antigens specific to the strain, which has so far prevented their specific identification.

Although the number of studies and of animal models developed is high, the antigens used have not induced sufficient protective immunity to lead to the development of human vaccines. In the case of Chlamydia pneumoniae, the role of the immune defense in the physiology and pathology of the disease should probably be understood in order to develop satisfactory vaccines.

More detailed information relating to the biology of these strains, their interactions with their hosts, the associated phenomena of infectivity and those of escaping the immune defenses of the host in particular, and finally their involvement in the development of the these associated pathologies, will allow a better understanding of these mechanisms. In the light of the preceding text which shows in particular the limitations of the means of controlling Chlamydia pneumoniae infection, it is therefore at present essential, on the one hand, to develop molecular tools, in particular from a better genetic knowledge of Chlamydia pneumoniae, but also to develop new preventive and therapeutic treatments, new diagnostic methods and new vaccine strategies which are specific, effective and tolerated. This is precisely the object of the present invention.

The subject of the present invention is the nucleotide sequence having the sequence SEQ ID No. 1 of the Chlamydia pneumoniae genome. However, the invention is not limited to SEQ ID No. 1, but encompasses genomes and nucleotides encoding polypeptides of strain variants, polymorphisms, allelic variants, and mutants.

Thus, the subject of the present invention encompasses nucleotide sequences characterized in that they are chosen from:

a) the nucleotide sequence of SEQ ID No. 1, a nucleotide sequence exhibiting at least 99.9% identity with the sequence SEQ ID No. 1, the nucleotide sequence of the genomic DNA contained within ATCC Deposit No. VR2634, the nucleotide sequence of a clone insert within ATCC Deposit No. 207000: 207001 and 207002;

b) a nucleotide sequence homologous to the sequence SEQ ID No. 1;

c) a polynucleotide sequence that hybridizes to the nucleotide sequence of a) under conditions of high or intermediate stringency as described below:

(i) By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C., the preferred hybridization temperature, in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Alternatively, the hybridization step can be performed at 65° C. in the presence of SSC buffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes can be performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and 0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. Other conditions of high stringency which may be used are well known in the art and as cited in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp. 9.47-9.57; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. are incorporated herein in their entirety.

(ii) By way of example and not limitation, procedures using conditions of intermediate stringency are as follows: Filters containing DNA are prehybridized, and then hybridized at a temperature of 60° C. in the presence of a 5×SSC buffer and labeled probe. Subsequently, filters washes are performed in a solution containing 2×SSC at 50° C. and the hybridized probes are detectable by autoradiography. Other conditions of intermediate stringency which may be used are well known in the art and as cited in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp. 9.47-9.57; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. are incorporated herein in their entirety.

d) a nucleotide sequence complementary to the sequence SEQ ID No. 1 or complementary to a nucleotide sequence as defined in a), b) or c) and a nucleotide sequence of their corresponding RNA;

e) a nucleotide sequence of a representative fragment of the sequence SEQ ID No. 1, or of a representative fragment of the nucleotide sequence as defined in a), b), c) or d);

f) a nucleotide sequence comprising a sequence as defined in a), b), c), d) or e);

g) a nucleotide sequence capable of being obtained from a nucleotide sequence as defined in a), b), c), d), e) or f); and

h) a modified nucleotide sequence of a nucleotide sequence as defined in a), b), c), d), e), f) or g).

Nucleotide sequence, polynucleotide or nucleic acid are understood to mean, according to the present invention, either a double-stranded DNA, a single-stranded DNA or products of transcription of the said DNAs.

It should be understood that the present invention does not relate to the genomic nucleotide sequences of Chlamydia pneumoniae taken in their natural environment, that is to say in the natural state. They are sequences which may have been isolated, purified or partially purified, by separation methods such as, for example, ion-exchange chromatography, molecular size exclusion chromatography or affinity chromatography, or alternatively fractionation techniques based on solubility in various solvents, or by genetic engineering methods such as amplification, cloning or subcloning, it being possible for the sequences of the invention to be carried by vectors.

The nucleotide sequence SEQ ID No. 1 was obtained by sequencing the Chlamydia pneumoniae genome by the method of directed sequencing after fluorescent automated sequencing of the inserts of clones and assembling of these sequences of nucleotide fragments (inserts) by means of softwares (cf. Examples). In spite of the high precision of the sequence SEQ ID No. 1, it is possible that it does not perfectly, 100% represent the nucleotide sequence of the Chlamydia pneumoniae genome and that a few rare sequencing errors or uncertainties still remain in the sequence SEQ ID No. 1. In the present invention, the presence of an uncertainty for an amino acid is designated by “Xaa” and that for a nucleotide is designated by “N” in the sequence listing below. These few rare errors or uncertainties could be easily detected and corrected by persons skilled in the art using the entire chromosome and/or its representative fragments according to the invention and standard amplification, cloning and sequencing methods, it being possible for the sequences obtained to be easily compared, in particular by means of a computer software and using computer-readable media for recording the sequences according to the invention as described, for example, below. After correcting these possible rare errors or uncertainties, the corrected nucleotide sequence obtained would still exhibit at least 99.9% identity with the sequence SEQ ID No. 1. Such rare sequencing uncertainties are not present within the DNA contained within ATCC Deposit No. VR2634, 207000, 207001 or 207002, and whatever rare sequence uncertainties that exist within SEQ ID No. 1 can routinely be corrected utilizing the DNA of the ATCC deposits.

Homologous nucleotide sequence for the purposes of the present invention is understood to mean a nucleotide sequence having a percentage identity with the bases of the nucleotide sequence SEQ ID No. 1 of at least 80%, preferably 90% and 95%, this percentage being purely statistical and it being possible for the differences between the two nucleotide sequences to be distributed randomly and over their entire length. The said homologous sequences exhibiting a percentage identity with the bases of the nucleotide sequence SEQ ID No. 1 of at least 80%, preferably 90% and 95%, may comprise, for example, the sequences corresponding to the genomic sequence or to the sequences of its representative fragments of a bacterium belonging to the Chlamydia family, including the species Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pecorum mentioned above, as well as the sequences corresponding to the genomic sequence or to the sequences of its representative fragments of a bacterium belonging to the variants of the species Chlamydia pneumoniae. In the present invention, the terms family and genus are mutually interchangeable, the terms variant, serotype, strain and subspecies are also mutually interchangeable. These homologous sequences may thus correspond to variations linked to mutations within the same species or between species and may correspond in particular to truncations, substitutions, deletions and/or additions of at least one nucleotide. The said homologous sequences may also correspond to variations linked to the degeneracy of the genetic code or to a bias in the genetic code which is specific to the family, to the species or to the variant and which are likely to be present in Chlamydia.

Protein and/or nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Thompson et al., 1994, Nucleic Acids Res. 22(2):4673-4680; Higgins et al., 1996, Methods Enzymol. 266:383-402; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Altschul et al., 1993, Nature Genetics 3:266-272).

In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”) which is well known in the art (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268; Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1993, Nature Genetics 3:266-272; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402). In particular, five specific BLAST programs are used to perform the following task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;

(3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database;

(4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and

(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992, Science 256:1443-1445; Henikoff and Henikoff, 1993, Proteins 17:49-61). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).

The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268).

Nucleotide sequence complementary to a sequence of the invention is understood to mean any DNA whose nucleotides are complementary to those of the sequence of the invention, and whose orientation is reversed (antiparallel sequence).

The present invention further comprises fragments of the sequences of a) through h), above. Representative fragments of the sequences according to the invention will be understood to mean any nucleotide fragment having at least 8 successive nucleotides, preferably at least 12 successive nucleotides, and still more preferably at least 15 or at least 20 successive nucleotides of the sequence from which it is derived. It is understood that such fragments refer only to portions of SEQ ID No. 1 that are not currently listed in a publicly available database.

Among these representative fragments, those capable of hybridizing under stringent conditions with a nucleotide sequence according to the invention are preferred. Hybridization under stringent conditions means that the temperature and ionic strength conditions are chosen such that they allow hybridization to be maintained between two complementary DNA fragments.

By way of illustration, high stringency conditions for the hybridization step for the purposes of defining the nucleotide fragments described above, are advantageously the following.

The hybridization is carried out at a preferred temperature of 65° C. in the presence of SSC buffer, 1×SSC corresponding to 0.15 M NaCl and 0.05 M Na citrate. The washing steps may be, for example, the following: 2×SSC, 0.1% SDS at room temperature followed by three washes with 1×SSC, 0.1% SDS; 0.5×SSC, 0.1% SDS; 0.1×SSC, 0.1% SDS at 68° C. for 15 minutes.

Intermediate stringency conditions, using, for example, a temperature of 60° C. in the presence of a 5×SSC buffer, or of low stringency, for example a temperature of 50° C. in the presence of a 5×SSC buffer, respectively require a lower overall complementarity for the hybridization between the two sequences.

The stringent hybridization conditions described above for a polynucleotide of about 300 bases in size will be adapted by persons skilled in the art for larger- or smaller-sized oligonucleotides, according to the teaching of Sambrook et al., 1989.

Among the representative fragments according to the invention, those which can be used as primer or probe in methods which make it possible to obtain homologous sequences or their representative fragments according to the invention, or to reconstitute a genomic fragment found to be incomplete in the sequence SEQ ID No. 1 or carrying an error or an uncertainty, are also preferred, these methods, such as the polymerase chain reaction (PCR), cloning and sequencing of nucleic acid being well known to persons skilled in the art. These homologous nucleotide sequences corresponding to mutations or to inter- or intra-species variations, as well as the complete genomic sequence or one of its representative fragments capable of being reconstituted, of course form part of the invention.

Among the said representative fragments, those which can be used as primer or probe in methods allowing diagnosis of the presence of Chlamydia pneumoniae or one of its associated microorganisms as defined below are also preferred.

The representative fragments capable of modulating, regulating, inhibiting or inducing the expression of a gene of Chlamydia pneumoniae or one of its associated microorganisms, and/or capable of modulating the replication cycle of Chlamydia pneumoniae or one of its associated microorganisms in the host cell and/or organism, are also preferred. Replication cycle is intended to designate invasion, multiplication, intracellular localization, in particular retention in the vacuole and inhibition of the process of fusion to the lysosome, and propagation of Chlamydia pneumoniae or one of its associated microorganisms from host cells to host cells.

Among the said representative fragments, those corresponding to nucleotide sequences corresponding to open reading frames, called ORF sequences (ORF for open reading frame), and encoding polypeptides, such as for example, but without being limited thereto, the ORF sequences which will be later described, are finally preferred.

The representative fragments according to the invention may be obtained, for example, by specific amplification, such as PCR, or after digestion, with appropriate restriction enzymes, of nucleotide sequences according to the invention; these methods are in particular described in the manual by Sambrook et al., 1989. The said representative fragments may also be obtained by chemical synthesis when they are not too large in size and according to methods well known to persons skilled in the art. For example, such fragments can be obtained by isolating fragments of the genomic DNA of ATCC Deposit No. VR2634 or a clone insert present at this ATCC Deposit No. 207000: 207001 and 207002.

The representative fragments according to the invention may be used, for example, as primer, to reconstitute some of the said representative fragments, in particular those in which a portion of the sequence is likely to be missing or imperfect, by methods well known to persons skilled in the art such as amplification, cloning or sequencing techniques.

Modified nucleotide sequence will be understood to mean any nucleotide sequence obtained by mutagenesis according to techniques well known to persons skilled in the art, and exhibiting modifications in relation to the normal sequences, for example mutations in the regulatory and/or promoter sequences for the expression of a polypeptide, in particular leading to a modification of the level of expression of the said polypeptide or to a modulation of the replicative cycle.

Modified nucleotide sequence will also be understood to mean any nucleotide sequence encoding a modified polypeptide as defined below.

The subject of the present invention also includes Chlamydia pneumoniae nucleotide sequences characterized in that they are chosen from a nucleotide sequence of an open reading frame (ORF), that is, the ORF2 to ORF1297 sequences.

The ORF2 to ORF1297 nucleotide sequences are defined in Tables 1 and 2, infra, by their position on the sequence SEQ ID No. 1. For example, the ORF2 sequence is defined by the nucleotide sequence between the nucleotides at position 42 and 794 on the sequence SEQ ID No. 1, ends included. ORF2 to ORF1297 have been identified via homology analyses as well as via analyses of potential ORF start sites, as discussed in the examples below. It is to be understood that each identified ORF of the invention comprises a nucleotide sequence that spans the contiguous nucleotide sequence from the codon immediately 3′ to the stop codon of the preceding ORF and through the 5′ codon to the next stop codon of SEQ ID No.:1 in-frame to the ORF nucleotide sequence. Table 2, infra, lists the beginning, end and potential start site of each of ORFs 1-1297. In one embodiment, the ORF comprises the contiguous nucleotide sequence spanning from the potential ORF start site downstream (that is, 3′) to the ORF stop codon (or the ORF codon immediately adjacent to and upstream of the ORF stop codon). ORF2 to ORF1297 encode the polypeptides of SEQ ID No. 2 to SEQ ID No. 1291 and of SEQ ID No. 6844 to SEQ ID No. 6849, respectively.

Upon introduction of minor frameshifts, certain individual ORFs can comprise larger “combined” ORFs. A list of such putative “combined” ORFs is shown in Table 3, below. For example, a combined ORF can comprise ORF 25, ORF 26 and ORF 27, including intervening in-frame, nucleotide sequences. The order of ORFs (5′ to 3′), within each “combined” ORF is as listed. It is to be understood that when ORF2 to ORF1297 are referred to herein, such reference is also meant to include “combined” ORFs. Polypeptide sequences encoded by such “combined” ORFs are also part of the present invention.

TABLE 3 ORF 25, ORF 26, ORF 27; ORF 28, ORF 29, ORF 30; ORF 31, ORF 32; ORF 33, ORF 35; ORF 466, ORF 467; ORF 468, ORF 469; ORF 477, ORF 476, ORF 474; ORF 480, ORF 482; ORF 483, ORF 485, ORF 486, ORF 500; ORF 503, ORF 504, ORF 505; ORF 506, ORF 507; ORF 1211, ORF 647; ORF 1286, ORF 1039; ORF 691, ORF 690; ORF 105, ORF 106; ORF 170, ORF 171; ORF 394, ORF 393; ORF 453, ORF 452, ORF 451; ORF 526, ORF 525; ORF 757, ORF 756, ORF 755; ORF 856, ORF 855; CRF 958, ORF 957; ORF 915, ORF 914, ORF 913; ORF 543, ORF 544; ORF 1266, ORF 380; ORF 745, ORF 744; ORF 777, ORF 776; ORF 343, ORF 1297, and representative fragments.

Table 1 also depicts the results of homology searches that compared the sequences of the polypeptides encoded by each of the ORFs to sequences present in public published databases. It is understood that those polypeptides listed in Table 1 as exhibiting greater than about 95% identity to a polypeptide present in a publicly disclosed database are not considered part of the present invention; likewise in this embodiment, those nucleotide sequences encoding such polypeptides are not considered part of the invention. In another embodiment, it is understood that those polypeptides listed in Table 1 as exhibiting greater than about 99% identity to a polypeptide present in a publicly disclosed database are not considered part of the invention; likewise, in this embodiment, those nucleotide sequences encoding such polypeptides are not considered part of the invention.

The invention also relates to the nucleotide sequences characterized in that they comprise a nucleotide sequence chosen from:

a) an ORF2 to ORF1297, a “combined” ORF nucleotide sequence, the nucleotide sequence of the genomic DNA contained within ATCC Deposit No. VR2634 or the nucleotide sequence of a clone insert in ATCC Deposit No. 207000: 207001 and 207002 according to the invention;

b) a homologous nucleotide sequence exhibiting at least 80% identity across an entire ORF2 to ORF1297 nucleotide sequence according to the invention or as defined in a);

c) a polynucleotide sequence that hybridizes to ORF2 to ORF1297 under conditions of high or intermediate stringency as described below:

(i) By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C., the preferred hybridization temperature, in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Alternatively, the hybridization step can be performed at 65° C. in the presence of SSC buffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes can be performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and 0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. Other conditions of high stringency which may be used are well known in the art and as cited in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp. 9.47-9.57; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. are incorporated herein in their entirety. Preferably, such sequences encode a homolog of a polypeptide encoded by one of ORF2 to ORF1297. In one embodiment, such sequences encode a Chlamydia pneumoniae polypeptide.

(ii) By way of example and not limitation, procedures using conditions of intermediate stringency are as follows: Filters containing DNA are prehybridized, and then hybridized at a temperature of 60° C. in the presence of a 5×SSC buffer and labeled probe. Subsequently, filters washes are performed in a solution containing 2×SSC at 50° C. and the hybridized probes are detectable by autoradiography. Other conditions of intermediate stringency which may be used are well known in the art and as cited in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp. 9.47-9.57; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. are incorporated herein in their entirety. Preferably, such sequences encode a homolog of a polypeptide encoded by one of ORF2 to ORF1297. In one embodiment, such sequences encode a Chlamydia pneumoniae polypeptide.

d) complementary or RNA nucleotide sequence corresponding to an ORF2 to ORF1297 sequence according to the invention or as defined in a), b) or c);

e) a nucleotide sequence of a representative fragment of an ORF2 to ORF1297 sequence according to the invention or of a sequence as defined in a), b), c) or d);

f) a nucleotide sequence capable of being obtained from an ORF2 to ORF1297 sequence according to the invention or as defined in a), b), c), d) or e); and

g) a modified nucleotide sequence of an ORF2 to ORF1297 sequence according to the invention or as defined in a), b), c), d), e) or f);

As regards the homology with the ORF2 to ORF1297 nucleotide sequences, the homologous sequences exhibiting a percentage identity with the bases of one of the ORF2 to ORF1297 nucleotide sequences of at least 80%, preferably 90% and 95%, are preferred. Such homologous sequences are identified routinely via, for example, the algorithms described above and in the examples below. The said homologous sequences correspond to the homologous sequences as defined above and may comprise, for example, the sequences corresponding to the ORF sequences of a bacterium belonging to the Chlamydia family, including the species Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pecorum mentioned above, as well as the sequences corresponding to the ORF sequences of a bacterium belonging to the variants of the species Chlamydia pneumoniae. These homologous sequences may likewise correspond to variations linked to mutations within the same species or between species and may correspond in particular to truncations, substitutions, deletions and/or additions of at least one nucleotide. The said homologous sequences may also correspond to variations linked to the degeneracy of the genetic code or to a bias in the genetic code which is specific to the family, to the species or to the variant and which are likely to be present in Chlamydia.

The invention comprises polypeptides encoded by a nucleotide sequence according to the invention, preferably by a representative fragment of the sequence SEQ ID No. 1 and corresponding to an ORF sequence, in particular the Chlamydia pneumoniae polypeptides, characterized in that they are chosen from the sequences SEQ ID No. 2 to SEQ ID No. 1291 or SEQ ID No. 6844 to SEQ ID No. 6849 and representative fragments thereof. However, the invention is not limited to polypeptides encoded by ORFs in SEQ ID No. 1 and its corresponding ORF sequences, but encompasses polypeptides of strain variants, polymorphisms, allelic variants, and mutants.

Thus, the invention also comprises the polypeptides characterized in that they comprise a polypeptide chosen from:

a) a polypeptide encoded by a polynucleotide sequence in SEQ ID No. 1 (e.g., any polypeptide encoded by a polynucleotide sequence corresponding to ORF2 to ORF1297 and/or representative fragments thereof) according to the invention;

b) a polypeptide homologous to a polypeptide according to the invention, or as defined in a);

c) a polypeptide encoded by a polynucleotide sequence that hybridizes to SEQ ID No. 1 or ORF2 to ORF1297 under high or intermediate stringency as described below:

(i) By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C., the preferred hybridization temperature, in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Alternatively, the hybridization step can be performed at 65° C. in the presence of SSC buffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes can be performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and 0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. Other conditions of high stringency which may be used are well known in the art and as cited in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp. 9.47-9.57; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. are incorporated herein in their entirety. Preferably such polypeptide represents a homolog of a polypeptide encoded by ORF2 to ORF1297. Preferably, such sequences encode a homolog of a polypeptide encoded by one of ORF2 to ORF1297. In one embodiment, such sequences encode a Chlamydia pneumoniae polypeptide.

(ii) By way of example and not limitation, procedures using conditions of intermediate stringency are as follows: Filters containing DNA are prehybridized, and then hybridized at a temperature of 60° C. in the presence of a 5×SSC buffer and labeled probe. Subsequently, filters washes are performed in a solution containing 2×SSC at 50° C. and the hybridized probes are detectable by autoradiography. Other conditions of intermediate stringency which may be used are well known in the art and as cited in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., pp. 9.47-9.57; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. are incorporated herein in their entirety. Preferably, such sequences encode a homolog of a polypeptide encoded by one of ORF2 to ORF1297. In one embodiment, such sequences encode a Chlamydia pneumoniae polypeptide.

d) a fragment of at least 5 amino acids of a polypeptide according to the invention, or as defined in a), b) or c);

e) a biologically active fragment of a polypeptide according to the invention, or as defined in a), b), c) or d); and

f) a modified polypeptide of a polypeptide according to the invention, as defined in a), b), c), d) or e).

In the present description, the terms polypeptide, peptide and protein are interchangeable.

It should be understood that the invention does not relate to the polypeptides in natural form, that is to say hat they are not taken in their natural environment but that they may have been isolated or obtained by purification from natural sources, or alternatively obtained by genetic recombination, or else by chemical synthesis and that they may, in this case, comprise nonnatural amino acids, as will be described below.

Homologous polypeptide will be understood to designate the polypeptides exhibiting, in relation to the natural polypeptide, certain modifications such as in particular a deletion, addition or substitution of at least one amino acid, a truncation, an extension, a chimeric fusion, and/or a mutation, or polypeptides exhibiting post-translational modifications. Among the homologous polypeptides, those whose amino acid sequence exhibits at least 80%, preferably 90%, homology or identity with the amino acid sequences of the polypeptides according to the invention are preferred. In the case of a substitution, one or more consecutive or nonconsecutive amino acids are replaced by “equivalent” amino acids. The expression “equivalent” amino acid is intended here to designate any amino acid capable of being substituted for one of the amino acids in the basic structure without, however, essentially modifying the biological activities of the corresponding peptides and as will be defined later.

Protein and/or nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Thompson et al., 1994, Nucleic Acids Res. 22(2):4673-4680; Higgins et al., 1996, Methods Enzymol. 266:383-402; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Altschul et al., 1993, Nature Genetics 3:266-272).

In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”) which is well know in the art (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268; Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1993, Nature Genetics 3:266-272; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402). In particular, five specific BLAST programs are used to perform the following task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;

(3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database;

(4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and

(5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992, Science 256:1443-1445; Henikoff and Henikoff, 1993, Proteins 17:49-61). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).

The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268).

Equivalent amino acids may be determined either based on their structural homology with the amino acids for which they are substituted, or on results of comparative tests of biological activity between the various polypeptides which may be carried out.

By way of example, there may be mentioned the possibilities of substitutions which may be carried out without resulting in a substantial modification of the biological activity of the corresponding modified polypeptides; the replacements, for example, of leucine with valine or isoleucine, of aspartic acid with glutamic acid, of glutamine with asparagine, of arginine with lysine, and the like, the reverse substitutions naturally being feasible under the same conditions.

The homologous polypeptides also correspond to the polypeptides encoded by the homologous nucleotide sequences as defined above and thus comprise in the present definition the mutated polypeptides or polypeptides corresponding to inter- or intra-species variations which may exist in Chlamydia, and which correspond in particular to truncations, substitutions, deletions and/or additions of at least one amino acid residue.

Biologically active fragment of a polypeptide according to the invention will be understood to designate in particular a polypeptide fragment, as defined below, exhibiting at least one of the characteristics of the polypeptides according to the invention, in particular in that it is:

capable of eliciting an immune response directed against Chlamydia pneumoniae; and/or

capable of being recognized by an antibody specific for a polypeptide according to the invention; and/or

capable of binding to a polypeptide or to a nucleotide sequence of Chlamydia pneumoniae; and/or

capable of modulating, regulating, inducing or inhibiting the expression of a gene of Chlamydia pneumoniae or one of its associated microorganisms, and/or capable of modulating the replication cycle of Chlamydia pneumoniae or one of its associated microorganisms in the host cell and/or organism; and/or

capable of generally exerting an even partial physiological activity, such as for example a structural activity (cellular envelope, ribosome), an enzymatic (metabolic) activity, a transport activity, an activity in the secretion or in the virulence.

A polypeptide fragment according to the invention is understood to designate a polypeptide comprising a minimum of 5 amino acids, preferably 10 amino acids or preferably 15 amino acids. It is to be understood that such fragments refer only to portions of polypeptides encoded by ORF2 to ORF1297 that are not currently listed in a publicly available database.

The polypeptide fragments according to the invention may correspond to isolated or purified fragments which are naturally present in Chlamydia pneumoniae or which are secreted by Chlamydia pneumoniae, or may correspond to fragments capable of being obtained by cleaving the said polypeptide with a proteolytic enzyme, such as trypsin or chymotrypsin or collagenase, or with a chemical reagent, such as cyanogen bromide (CNBr) or alternatively by placing the said polypeptide in a highly acidic environment, for example at pH 2.5. Such polypeptide fragments may be equally well prepared by chemical synthesis, using hosts transformed with an expression vector according to the invention containing a nucleic acid allowing the expression of the said fragments, placed under the control of appropriate elements for regulation and/or expression.

“Modified polypeptide” of a polypeptide according to the invention is understood to designate a polypeptide obtained by genetic recombination or by chemical synthesis as will be described below, exhibiting at least one modification in relation to the normal sequence. These modifications may in particular affect amino acids responsible for a specificity or for the efficiency of the activity, or responsible for the structural conformation, for the charge or for the hydrophobicity, and for the capacity for multimerization and for membrane insertion of the polypeptide according to the invention. It is thus possible to create polypeptides with an equivalent, an increased or a reduced activity, and with an equivalent, a narrower or a broader specificity. Among the modified polypeptides, there may be mentioned the polypeptides in which up to 5 amino acids may be modified, truncated at the N- or C-terminal end, or alternatively deleted, or else added.

As is indicated, the modifications of the polypeptide may have in particular the objective:

of making it capable of modulating, regulating, inhibiting or inducing the expression of a gene of Chlamydia, in particular of Chlamydia pneumoniae and its variants, or one of its associated microorganisms, and/or capable of modulating the replication cycle of Chlamydia, in particular of Chlamydia pneumoniae and its variants, or one of its associated microorganisms, in the host cell and/or organism,

of allowing its use in methods of biosynthesis or of biodegradation, or its incorporation into vaccine compositions,

of modifying its bioavailability as a compound for therapeutic use.

The said modified polypeptides may also be used on any cell or microorganism for which the said modified polypeptides will be capable of modulating, regulating, inhibiting or inducing gene expression, or of modulating the growth or the replication cycle of the said cell or of the said microorganism. The methods allowing demonstration of the said modulations on eukaryotic or prokaryotic cells are well known to persons skilled in the art. The said cells or microorganisms will be chosen, in particular, from tumour cells or infectious microorganisms and the said modified polypeptides may be used for the prevention or treatment of pathologies linked to the presence of the said cells or of the said microorganisms. It is also clearly understood that the nucleotide sequences encoding the said modified polypeptides may be used for the said modulations, for example by the intermediacy of vectors according to the invention and which are described below, so as to prevent or to treat the said pathologies.

The above modified polypeptides may be obtained using combinatory chemistry, in which it is possible to systematically vary portions of the polypeptide before testing them on models, cell cultures or microorganisms for example, so as to select the compounds which are the most active or which exhibit the desired properties.

Chemical synthesis also has the advantage of being able to use:

nonnatural amino acids, or

nonpeptide bonds.

Accordingly, in order to extend the life of the polypeptides according to the invention, it may be advantageous to use nonnatural amino acids, for example in the D form, or alternatively amino acid analogues, in particular sulphur-containing forms for example.

Finally, the structure of the polypeptides according to the invention, its homologous or modified forms, as well as the corresponding fragments may be integrated into chemical structures of the polypeptide type and the like. Accordingly, it may be advantageous to provide at the N- and C-terminal ends compounds which are not recognized by proteases.

Also forming part of the invention are the nucleotide sequences encoding a polypeptide according to the invention. Described below are ORF nucleotide sequences encoding polypeptides exhibiting particularly preferable characteristics. For each group of preferred ORFS described below, it is to be understood that in addition to the individual ORFs listed, in instances wherein such ORFS are present as part of “combined” ORFs, the “combined” ORFs are also to be included within the preferred group.

More particularly, the subject of the invention is nucleotide sequences, characterized in that they encode a polypeptide of the cellular envelope, preferably of the outer cellular envelope of Chlamydia pneumoniae or one of its representative fragments, such as for example the predominant proteins of the outer membrane, the adhesion proteins or the proteins entering into the composition of the Chlamydia wall. Among these sequences, the sequences comprising a nucleotide sequence chosen from the following sequences are most preferred:

ORF15; ORF25; ORF26; ORF27; ORF28; ORF29; ORF30; ORF31; ORF32; ORF33; ORF35; ORF68; ORF124; ORF275; ORF291; ORF294; ORF327; ORF342; ORF364; ORF374; ORF380; ORF414; ORF439; ORF466; ORF467; ORF468; ORF469; ORF470; ORF472; ORF474; ORF476; ORF477; ORF478; ORF479; ORF480; ORF482; ORF485; ORF500; ORF501; ORF503; ORF504; ORF505; ORF506; ORF520; ORF578; ORF580; ORF581; ORF595; ORF596; ORF597; ORF737; ORF830; ORF834; ORF836; ORF893; ORF917; ORF932; ORF976; ORF1035; ORF1045; ORF1090 and one of their representative fragments.

The structure of the cytoplasmic membranes and of the wall of bacteria is dependent on the associated proteins. The structure of the cytoplasmic membrane makes it impermeable to water, to water-soluble substances and to small-sized molecules (ions, small inorganic molecules, peptides or proteins). To enter into or to interfere with a cell or a bacterium, a ligand must establish a special relationship with a protein anchored in the cytoplasmic membrane (the receptor). These proteins which are anchored on the membrane play an important role in metabolism since they control the exchanges in the bacterium. These exchanges apply to molecules of interest for the bacterium (small molecules such as sugars and small peptides) as well as undesirable molecules for the bacterium such as antibiotics or heavy metals.

The double lipid layer structure of the membrane requires the proteins which are inserted therein to have hydrophobic domains of about twenty amino acids forming an alpha helix. Predominantly hydrophobic and potentially transmembrane regions may be predicted from the primary sequence of the proteins, itself deduced from the nucleotide sequence. The presence of one or more putative transmembrane domains raises the possibility for a protein to be associated with the cytoplasmic membrane and to be able to play an important metabolic role therein or alternatively for the protein thus exposed to be able to exhibit potentially protective epitopes.

If the proteins inserted into the membrane exhibit several transmembrane domains capable of interacting with one another via electrostatic bonds, it then becomes possible for these proteins to form pores which go across the membrane which becomes permeable for a number of substances. It should be noted that proteins which do not have transmembrane domains may also be anchored by the intermediacy of fatty acids in the cytoplasmic membrane, it being possible for the breaking of the bond between the protein and its anchor in some cases to be responsible for the release of the peptide outside the bacterium.

Preferably, the invention relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae transmembrane polypeptide or one of its representative fragments, having between 1 and 3 transmembrane domains and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF2; ORF3; ORF6; ORF9; ORF10; ORF11; ORF13; ORF14; ORF16; ORF18; ORF19; ORF20; ORF21; ORF22; ORF25; ORF27; ORF28; ORF29; ORF30; ORF31; ORF32; ORF33; ORF34; ORF35; ORF37; ORF39; ORF41; ORF42; ORF44; ORF45; ORF46; ORF47; ORF48; ORF49; ORF50; ORF53; ORF54; ORF56; ORF57; ORF59; ORF60; ORF61; ORF62; ORF63; ORF64; ORF65; ORF66; ORF69; ORF72; ORF73; ORF74; ORF76; ORF77; ORF78; ORF79; ORF80; ORF82; ORF84; ORF85; ORF86; ORF88; ORF89; ORF90; ORF91; ORF92; ORF93; ORF95; ORF96; ORF98; ORF99; ORF100; ORF101; ORF102; ORF103; ORF104; ORF105; ORF106; ORF107; ORF108; ORF114; ORF117; ORF118; ORF122; ORF123; ORF124; ORF125; ORF129; ORF130; ORF131; ORF132; ORF133; ORF134; ORF135; ORF137; ORF138; ORF139; ORF140; ORF141; ORF142; ORF143; ORF145; ORF146; ORF147; ORF150; ORF151; ORF152; ORF156; ORF157; ORF158; ORF159; ORF160; ORF161; ORF162; ORF164; ORF166; ORF167; ORF170; ORF173; ORF175; ORF176; ORF178; ORF179; ORF180; ORF182; ORF183; ORF184; ORF185; ORF186; ORF187; ORF188; ORF189; ORF190; ORF191; ORF192; ORF194; ORF195; ORF196; ORF197; ORF198; ORF199; ORF200; ORF201; ORF202; ORF205; ORF207; ORF208; ORF209; ORF210; ORF212; ORF215; ORF219; ORF220; ORF224; ORF226; ORF227; ORF228; ORF231; ORF232; ORF233; ORF234; ORF235; ORF236; ORF238; ORF239; ORF240; ORF241; ORF242; ORF244; ORF247; ORF251; ORF252; ORF253; ORF255; ORF256; ORF257; ORF258; ORF260; ORF262; ORF263; ORF266; ORF267; ORF268; ORF269; ORF270; ORF273; ORF274; ORF276; ORF278; ORF279; ORF280; ORF281; ORF282; ORF283; ORF284; ORF286; ORF287; ORF289; ORF290; ORF291; ORF293; ORF294; ORF297; ORF304; ORF305; ORF307; ORF308; ORF309; ORF310; ORF311; ORF313; ORF314; ORF315; ORF316; ORF318; ORF319; ORF320; ORF321; ORF322; ORF323; ORF324; ORF325; ORF326; ORF331; ORF332; ORF336; ORF338; ORF339; ORF341; ORF344; ORF345; ORF346; ORF350; ORF352; ORF353; ORF356; ORF357; ORF358; ORF359; ORF360; ORF362; ORF365; ORF366; ORF367; ORF370; ORF372; ORF373; ORF376; ORF377; ORF378; ORF379; ORF381; ORF382; ORF383; ORF384; ORF385; ORF386; ORF387; ORF390; ORF392; ORF393; ORF394; ORF396; ORF398; ORF399; ORF400; ORF404; ORF408; ORF410; ORF411; ORF413; ORF416; ORF417; ORF418; ORF420; ORF422; ORF424; ORF427; ORF428; ORF429; ORF430; ORF431; ORF433; ORF434; ORF437; ORF440; ORF441; ORF442; ORF443; ORF444; ORF445; ORF447; ORF450; ORF451; ORF452; ORF455; ORF456; ORF459; ORF460; ORF461; ORF462; ORF463; ORF464; ORF465; ORF467; ORF469; ORF471; ORF474; ORF475; ORF476; ORF477; ORF479; ORF482; ORF483; ORF484; ORF485; ORF486; ORF487; ORF488; ORF491; ORF493; ORF494; ORF497; ORF498; ORF499; ORF503; ORF508; ORF509; ORF510; ORF512; ORF514; ORF515; ORF516; ORF517; ORF518; ORF520; ORF521; ORF523; ORF525; ORF527; ORF528; ORF529; ORF530; ORF531; ORF533; ORF534; ORF535; ORF536; ORF537; ORF540; ORF541; ORF543; ORF544; ORF545; ORF546; ORF548; ORF549; ORF551; ORF553; ORF554; ORF555; ORF556; ORF557; ORF558; ORF559; ORF560; ORF562; ORF563; ORF564; ORF565; ORF566; ORF569; ORF571; ORF573; ORF576; ORF577; ORF581; ORF583; ORF584; ORF585; ORF586; ORF588; ORF591; ORF592; ORF594; ORF595; ORF596; ORF597; ORF599; ORF600; ORF603; ORF605; ORF608; ORF614; ORF615; ORF620; ORF621; ORF622; ORF623; ORF624; ORF625; ORF629; ORF630; ORF631; ORF633; ORF634; ORF637; ORF642; ORF644; ORF645; ORF647; ORF648; ORF652; ORF654; ORF655; ORF657; ORF658; ORF659; ORF660; ORF661; ORF664; ORF665; ORF666; ORF667; ORF670; ORF671; ORF672; ORF673; ORF674; ORF676; ORF679; ORF681; ORF684; ORF687; ORF688; ORF689; ORF690; ORF693; ORF694; ORF695; ORF696; ORF697; ORF698; ORF699; ORF700; ORF701; ORF703; ORF705; ORF706; ORF707; ORF708; ORF710; ORF712; ORF715; ORF716; ORF717; ORF718; ORF719; ORF721; ORF722; ORF723; ORF725; ORF726; ORF727; ORF728; ORF729; ORF730; ORF731; ORF733; ORF736; ORF737; ORF738; ORF740; ORF741; ORF742; ORF743; ORF747; ORF748; ORF750; ORF752; ORF754; ORF755; ORF756; ORF757; ORF759; ORF760; ORF761; ORF762; ORF763; ORF764; ORF765; ORF766; ORF767; ORF768; ORF772; ORF774; ORF775; ORF777; ORF781; ORF783; ORF788; ORF791; ORF792; ORF793; ORF794; ORF795; ORF796; ORF797; ORF798; ORF799; ORF802; ORF803; ORF806; ORF807; ORF808; ORF809; ORF810; ORF811; ORF813; ORF814; ORF815; ORF816; ORF817; ORF819; ORF820; ORF821; ORF823; ORF824; ORF827; ORF829; ORF830; ORF831; ORF833; ORF834; ORF835; ORF837; ORF844; ORF845; ORF846; ORF847; ORF848; ORF849; ORF850; ORF851; ORF852; ORF854; ORF855; ORF856; ORF857; ORF859; ORF860; ORF862; ORF865; ORF866; ORF868; ORF869; ORF870; ORF871; ORF872; ORF874; ORF877; ORF878; ORF879; ORF880; ORF881; ORF882; ORF884; ORF885; ORF888; ORF889; ORF890; ORF891; ORF892; ORF894; ORF895; ORF896; ORF897; ORF899; ORF900; ORF902; ORF903; ORF904; ORF905; ORF909; ORF910; ORF912; ORF913; ORF914; ORF915; ORF917; ORF918; ORF919; ORF921; ORF923; ORF924; ORF926; ORF927; ORF928; ORF929; ORF930; ORF931; ORF937; ORF938; ORF939; ORF941; ORF943; ORF948; ORF951; ORF952; ORF953; ORF958; ORF960; ORF963; ORF964; ORF965; ORF968; ORF970; ORF974; ORF975; ORF977; ORF979; ORF980; ORF981; ORF983; ORF984; ORF985; ORF987; ORF989; ORF992; ORF993; ORF997; ORF998; ORF999; ORF1001; ORF1002; ORF1004; ORF1005; ORF1009; ORF1013; ORF1014; ORF1015; ORF1016; ORF1019; ORF1021; ORF1023; ORF1024; ORF1029; ORF1031; ORF1033; ORF1034; ORF1039; ORF1041; ORF1042; ORF1045; ORF1047; ORF1049; ORF1051; ORF1052; ORF1053; ORF1054; ORF1056; ORF1059; ORF1061; ORF1062; ORF1063; ORF1064; ORF1065; ORF1067; ORF1075; ORF1077; ORF1078; ORF1079; ORF1080; ORF1081; ORF1089; ORF1095; ORF1097; ORF1098; ORF1099; ORF1101; ORF1102; ORF1103; ORF1106; ORF1107; ORF1108; ORF1109; ORF1110; ORF1113; ORF1116; ORF1118; ORF1119; ORF1121; ORF1123; ORF1124; ORF1126; ORF1128; ORF1130; ORF1131; ORF1133; ORF1134; ORF1136; ORF1137 and one of their representative fragments.

Preferably, the invention relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae transmembrane polypeptide or one of its representative fragments, having between 4 and 6 transmembrane domains and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF5; ORF7; ORF8; ORF15; ORF36; ORF38; ORF51; ORF55; ORF58; ORF67; ORF70; ORF81; ORF97; ORF110; ORF111; ORF115; ORF119; ORF126; ORF128; ORF148; ORF155; ORF163; ORF165; ORF168; ORF169; ORF171; ORF172; ORF174; ORF177; ORF181; ORF193; ORF203; ORF213; ORF214; ORF216; ORF217; ORF221; ORF222; ORF225; ORF229; ORF243; ORF246; ORF248; ORF254; ORF261; ORF285; ORF288; ORF292; ORF296; ORF298; ORF299; ORF301; ORF303; ORF317; ORF328; ORF329; ORF351; ORF354; ORF355; ORF364; ORF371; ORF374; ORF375; ORF391; ORF395; ORF401; ORF403; ORF405; ORF409; ORF414; ORF419; ORF421; ORF423; ORF425; ORF438; ORF448; ORF453; ORF458; ORF466; ORF468; ORF470; ORF480; ORF489; ORF490; ORF496; ORF501; ORF504; ORF505; ORF506; ORF511; ORF513; ORF519; ORF526; ORF532; ORF538; ORF539; ORF547; ORF550; ORF561; ORF568; ORF570; ORF574; ORF578; ORF579; ORF580; ORF582; ORF589; ORF593; ORF598; ORF601; ORF604; ORF610; ORF613; ORF617; ORF626; ORF632; ORF635; ORF638; ORF640; ORF641; ORF646; ORF649; ORF650; ORF651; ORF686; ORF711; ORF724; ORF732; ORF734; ORF744; ORF745; ORF749; ORF751; ORF769; ORF770; ORF771; ORF773; ORF776; ORF779; ORF780; ORF785; ORF787; ORF789; ORF801; ORF805; ORF812; ORF822; ORF825; ORF826; ORF839; ORF841; ORF843; ORF853; ORF861; ORF875; ORF876; ORF886; ORF893; ORF898; ORF906; ORF907; ORF908; ORF920; ORF922; ORF925; ORF933; ORF935; ORF936; ORF944; ORF946; ORF947; ORF954; ORF959; ORF961; ORF966; ORF967; ORF972; ORF978; ORF995; ORF996; ORF1000; ORF1003; ORF1010; ORF1011; ORF1012; ORF1017; ORF1020; ORF1030; ORF1036; ORF1038; ORF1043; ORF1046; ORF1048; ORF1050; ORF1058; ORF1071; ORF1073; ORF1084; ORF1085; ORF1086; ORF1087; ORF1091; ORF1092; ORF1094; ORF1096; ORF1100; ORF1104; ORF1111; ORF1112; ORF1114; ORF1117; ORF1122; ORF1125 and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae transmembrane polypeptide or one of its representative fragments, having at least 7 transmembrane domains and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF17; ORF52; ORF68; ORF83; ORF87; ORF109; ORF112; ORF113; ORF120; ORF121; ORF127; ORF153; ORF204; ORF211; ORF218; ORF223; ORF275; ORF277; ORF295; ORF300; ORF302; ORF306; ORF327; ORF335; ORF342; ORF343; ORF347; ORF349; ORF361; ORF363; ORF369; ORF380; ORF388; ORF389; ORF397; ORF415; ORF432; ORF439; ORF446; ORF449; ORF472; ORF478; ORF500; ORF522; ORF524; ORF567; ORF575; ORF602; ORF606; ORF609; ORF636; ORF639; ORF643; ORF653; ORF668; ORF692; ORF702; ORF704; ORF713; ORF720; ORF778; ORF784; ORF800; ORF836; ORF838; ORF842; ORF864; ORF867; ORF883; ORF901; ORF916; ORF932; ORF934; ORF940; ORF942; ORF950; ORF956; ORF971; ORF973; ORF976; ORF988; ORF994; ORF1018; ORF1028; ORF1035; ORF1037; ORF1044; ORF1055; ORF1057; ORF1068; ORF1069; ORF1070; ORF1072; ORF1082; ORF1088; ORF1105; ORF1132; ORF1135 and one of their representative fragments.

Preferably, the invention relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae surface exposed polypeptide (e.g., an outer membrane protein) or one of its representative fragments, said nucleotide sequences comprising a nucleotide sequence chosen from the following sequences:

ORF 15, ORF 25, ORF 26, ORF 27, ORF 28, ORF 29, ORF 30, ORF 31, ORF 32, ORF 33, ORF 35, ORF 36, ORF 1257, ORF 280, ORF 291, ORF 314, ORF 354, ORF 380, ORF 1266, ORF 466, ORF 467, ORF 468, ORF 469, ORF 470, ORF 472, ORF 474, ORF 476, ORF 477, ORF 478, ORF 479, ORF 480, ORF 482, ORF 483, ORF 485, ORF 486, ORF 500, ORF 501, ORF 503, ORF 504, ORF 505, ORF 506, ORF 507, ORF 1268, ORF 1269, ORF 543, ORF 544, ORF 578, ORF 579, ORF 580, ORF 581, ORF 595, ORF 596, ORF 597, ORF 1271, ORF 633, ORF 637, ORF 699, ORF 706, ORF 737, ORF 744, ORF 1273, ORF 751, ORF 775, ORF 776, ORF 777, ORF 793, ORF 815, ORF 830, ORF 1221, ORF 849, ORF 851, ORF 852, ORF 874, ORF 891, ORF 922, ORF 940, ORF 1231, ORF 1281, ORF 1035, ORF 1079, ORF 1087, ORF 1108, and one of their representative fragments.

Preferably, the invention relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae lipoprotein or one of its representative fragments, said nucleotide sequences comprising a nucleotide sequence chosen from the following sequences:

ORF 3, ORF 10, ORF 11, ORF 16, ORF 1254, ORF 1255, ORF 38, ORF 1256, ORF 62, ORF 85, ORF 1258, ORF 115, ORF 1151, ORF 151, ORF 1259, ORF 173, ORF 1261, ORF 186, ORF 194, ORF 205, ORF 214, ORF 216, ORF 217, ORF 238, ORF 1177, ORF 280, ORF 291, ORF 317, ORF 327, ORF 354, ORF 364, ORF 367, ORF 414, ORF 432, ORF 1192, ORF 460, ORF 1267, ORF 1268, ORF 520, ORF 536, ORF 1270, ORF 576, ORF 597, ORF 603, ORF 609, ORF 637, ORF 1272, ORF 652, ORF 1213, ORF 699, ORF 705, ORF 706, ORF 708, ORF 711, ORF 727, ORF 1274, ORF 800, ORF 814, ORF 825, ORF 829, ORF 830, ORF 831, ORF 844, ORF 849, ORF 1275, ORF 1276, ORF 1277, ORF 872, ORF 878, ORF 880, ORF 891, ORF 892, ORF 1278, ORF 1279, ORF 1280, ORF 941, ORF 942, ORF 1282, ORF 1283, ORF 952, ORF 988, ORF 998, ORF 1009, ORF 1285, ORF 1235, ORF 1028, ORF 1056, ORF 1070, ORF 1287, ORF 1087, ORF 1288, ORF 1289, ORF 1098, ORF 1246, ORF 1291, ORF 1108, ORF 1109, ORF 1112, ORF 1133, and one of their representative fragments.

Preferably, the invention relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae polypeptide involved in lipopolysaccharide (LPS) biosynthesis, said nucleotide sequences comprising a nucleotide sequence chosen from the following sequences: ORF 316, ORF 564, ORF 610, ORF 647, ORF 1211, ORF 688, ORF 924, and one of their representative fragments.

Preferably the invention relates to additional LPS-related nucleotide sequences according to the invention, characterized in that they encode:

(a) a Chlamydia pneumoniae KDO (3-deoxy-D-manno-octulosonic acid)-related polypeptide or one of its representative fragments, said nucleotide sequences comprising a nucleotide sequence chosen from the following sequences: ORF 177, ORF 1156, ORF 245, ORF 767, and one of their representative fragments;

(b) a Chlamydia pneumoniae phosphomannomutase-related polypeptide or one of its representative fragments, said nucleotide sequences comprising a nucleotide sequence chosen from the following sequences: ORF 74, and one of its representative fragments;

(c) a Chlamydia pneumoniae phosphoglucomutase-related polypeptide or one of its representative fragments, said nucleotide sequences comprising a nucleotide sequence chosen from the following sequences: ORF 1286, ORF 1039, and one of their representative fragments; and

(d) a Chlamydia pneumoniae lipid A component-related polypeptide or one of its representative fragments, said nucleotide sequences comprising a nucleotide sequence chosen from the following sequences: ORF 689, ORF 690, ORF 691, ORF 1037, and one of their representative fragments.

Preferably, the invention relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae polypeptide containing RGD (Arg-Gly-Asp) attachment sites or one of its representative fragments.

(a) RGD-containing proteins that are outer membrane proteins, are more likely to play a role in cell attachment. ORFs that encoded a protein containing an RGD sequence and also were classified as outer membrane proteins are ORF 468 and its representative fragments.

(a) An RGD-encoding ORF that showed homology to cds1, cds2, and copN type III virulence loci in Chlamydia psittaci (Hsia, R. et al. (1997), Type III secretion genes identity a putative virulence locus of Chlamydia. Molecular Microbiology 25:351-359) is ORF 350, and its representative fragments.

(c) The outer membrane of Chlamydia is made of cysteine-rich proteins that form a network of both intra and inter molecular disulfide links. This contributes to the integrity of the membrane since Chlamydia lacks the peptidoglycan layer that other gram-negative bacteria have. Cysteine-rich proteins that have the RGD sequence are also considered to be potential vaccine candidates. Cysteine-rich proteins were defined as proteins that had more than 3.0% cysteine in their primary amino acid sequence, above the mean genomic ORF cysteine content. The corresponding ORFs are: ORF 1290, ORF 1294, ORF 1296, and one of their representative fragments.

(d) The outer membrane of Chlamydia may also contain small proteins that have cysteines in their N- and C-terminus that may contribute to the network formed by disulfide linkages. These proteins may be anchored in the outer membrane via their N-terminus and may have their C-terminus exposed, which then can interact with the host cells. Alternatively, these proteins may be anchored in the outer membrane via both N-and C-terminus and may have regions in the middle that may be exposed which can in turn interact with the host cells. ORFs encoding polypeptides that contain cysteines in their first 30 amino acids and also contain an RGD sequence are:

ORF 105, ORF 106, ORF 114, ORF 170, ORF 171, ORF 1264, ORF 268, ORF 1265, ORF 350, ORF 393, ORF 394, ORF 451, ORF 452, ORF 453, ORF 473, ORF 499, ORF 515, ORF 519, ORF 525, ORF 526, ORF 538, ORF 611, ORF 645, ORF 686, ORF 700, ORF 746, ORF 755, ORF 756, ORF 757, ORF 789, ORF 814, ORF 855, ORF 856, ORF 878, ORF 957, ORF 958, ORF 989, ORF 1290, and one of their representative fragments.

(e) RGD-containing ORFs homologous to RGD-containing ORFs from Chlamydia trachomatis are:

ORF 114, ORF 468, ORF 755, ORF 756, ORF 757, ORF 855, ORF 856, ORF 905, ORF 913, ORF 914, ORF 915, and one of their representative fragments.

Preferably, the invention relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae Type III or other, non-type III secreted polypeptide or one of its representative fragments, said nucleotide sequences comprising a nucleotide sequence chosen from the following sequences:

ORF 25, ORF 28, ORF 29, ORF 33, ORF 308, ORF 309, ORF 343, ORF 344, ORF 345, ORF 367, ORF 414, ORF 415, ORF 480, ORF 550, ORF 579, ORF 580, ORF 581, ORF 597, ORF 699, ORF 744, ORF 751, ORF 776, ORF 866, ORF 874, ORF 883, ORF 884, ORF 888, ORF 891, ORF 1293, and one of their representative fragments.

Preferably, the invention relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae cell wall anchored surface polypeptide or one of its representative fragments, said nucleotide sequences comprising a nucleotide sequence chosen from the following sequences: ORF 267, ORF 271, ORF 419, ORF 590, ORF 932, ORF 1292, ORF 1295, and one of their representative fragments.

Preferably, the invention relates to the nucleotide sequences according to the invention, characterized in that they encode Chlamydia pneumoniae polypeptides not found in Chlamydia trachomatis (Blastp. P>e⁻¹⁰), said nucleotide sequences comprising a nucleotide sequence chosen from the following sequences:

ORF 7, ORF 8, ORF 9, ORF 16, ORF 17, ORF 18, ORF 19, ORF 20, ORF 21, ORF 22, ORF 1254, ORF 23, ORF 1255, ORF 24, ORF 1139, ORF 1140, ORF 46, ORF 47, ORF 51, ORF 60, ORF 1256, ORF 61, ORF 62, ORF 63, ORF 64, ORF 1257, ORF 65, ORF 66, ORF 67, ORF 68, ORF 1143, ORF 1145, ORF 83, ORF 84, ORF 1146, ORF 85, ORF 86, ORF 87, ORF 1258, ORF 116, ORF 117, ORF 125, ORF 1148, ORF 143, ORF 1150, ORF 1151, ORF 144, ORF 145, ORF 147, ORF 148, ORF 149, ORF 150, ORF 152, ORF 1259, ORF 162, ORF 166, ORF 1154, ORF 167, ORF 1261, ORF 1156, ORF 1157, ORF 178, ORF 179, ORF 1158, ORF 182, ORF 183, ORF 184, ORF 185, ORF 1159, ORF 186, ORF 1160, ORF 187, ORF 188, ORF 189, ORF 190, ORF 1161, ORF 1162, ORF 191, ORF 192, ORF 194, ORF 195, ORF 1163, ORF 196, ORF 201, ORF 202, ORF 209, ORF 212, ORF 221, ORF 224, ORF 1167, ORF 226, ORF 227, ORF 228, ORF 229, ORF 230, ORF 231, ORF 232, ORF 1169, ORF 1170, ORF 1171, ORF 234, ORF 235, ORF 236, ORF 1172, ORF 243, ORF 251, ORF 252, ORF 1176, ORF 253, ORF 255, ORF 254, ORF 256, ORF 1177, ORF 1178, ORF 262, ORF 263, ORF 1264, ORF 278, ORF 279, ORF 1180, ORF 280, ORF 290, ORF 291, ORF 292, ORF 296, ORF 1181, ORF 297, ORF 298, ORF 300, ORF 1265, ORF 322, ORF 324, ORF 325, ORF 370, ORF 1186, ORF 371, ORF 372, ORF 1187, ORF 373, ORF 378, ORF 1266, ORF 382, ORF 383, ORF 384, ORF 385, ORF 386, ORF 1188, ORF 1189, ORF 391, ORF 392, ORF 398, ORF 400, ORF 403, ORF 1191, ORF 423, ORF 435, ORF 445, ORF 450, ORF 1193, ORF 456, ORF 460, ORF 461, ORF 465, ORF 1196, ORF 471, ORF 473, ORF 475, ORF 481, ORF 484, ORF 487, ORF 488, ORF 489, ORF 490, ORF 491, ORF 492, ORF 493, ORF 494, ORF 495, ORF 496, ORF 497, ORF 498, ORF 499, ORF 502, ORF 1267, ORF 1268, ORF 508, ORF 510, ORF 509, ORF 512, ORF 515, ORF 519, ORF 1197, ORF 521, ORF 1198, ORF 522, ORF 524, ORF 528, ORF 534, ORF 537, ORF 1269, ORF 1270, ORF 548, ORF 551, ORF 557, ORF 1201, ORF 1203, ORF 562, ORF 566, ORF 593, ORF 595, ORF 600, ORF 1271, ORF 604, ORF 611, ORF 612, ORF 614, ORF 616, ORF 625, ORF 627, ORF 628, ORF 629, ORF 631, ORF 641, ORF 1272, ORF 648, ORF 1212, ORF 663, ORF 685, ORF 707, ORF 714, ORF 715, ORF 716, ORF 717, ORF 722, ORF 746, ORF 1273, ORF 761, ORF 764, ORF 770, ORF 1217, ORF 783, ORF 1274, ORF 803, ORF 815, ORF 1220, ORF 835, ORF 1221, ORF 844, ORF 845, ORF 846, ORF 847, ORF 848, ORF 849, ORF 850, ORF 851, ORF 1275, ORF 852, ORF 862, ORF 1276, ORF 1277, ORF 873, ORF 1223, ORF 892, ORF 919, ORF 1225, ORF 1278, ORF 926, ORF 1228, ORF 1229, ORF 1230, ORF 1279, ORF 1281, ORF 1282, ORF 1283, ORF 948, ORF 950, ORF 949, ORF 951, ORF 980, ORF 982, ORF 1233, ORF 999, ORF 1000, ORF 1001, ORF 1002, ORF 1008, ORF 1285, ORF 1235, ORF 1016, ORF 1019, ORF 1027, ORF 1036, ORF 1241, ORF 1048, ORF 1049, ORF 1050, ORF 1053, ORF 1054, ORF 1064, ORF 1076, ORF 1091, ORF 1288, ORF 1093, ORF 1289, ORF 1101, ORF 1103, ORF 1245, ORF 1246, ORF 1247, ORF 1290, ORF 1291, ORF 1115, ORF 1116, ORF 1118, ORF 1120, ORF 1249, ORF 1121, ORF 1250, ORF 1126, ORF 1251, ORF 1127, ORF 1128, ORF 1130, ORF 1129, ORF 1131, ORF 1136, ORF 1253, ORF 1292, ORF 1294, ORF 1295, ORF 1296, and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the intermediate metabolism, in particular in the metabolism of sugars and/or of cofactors, such as for example triose phosphate isomerase or pyruvate kinase, and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF2; ORF55; ORF56; ORF69; ORF75; ORF80; ORF100; ORF110; ORF114; ORF120; ORF121; ORF157; ORF160; ORF161; ORF172; ORF180; ORF181; ORF198; ORF200; ORF225; ORF248; ORF249; ORF276; ORF277; ORF318; ORF319; ORF320; ORF323; ORF331; ORF347; ORF375; ORF376; ORF381; ORF393; ORF394; ORF395; ORF396; ORF409; ORF446; ORF447; ORF448; ORF449; ORF513; ORF516; ORF571; ORF647; ORF662; ORF697; ORF718; ORF793; ORF794; ORF808; ORF809; ORF838; ORF839; ORF840; ORF853; ORF854; ORF918; ORF923; ORF929; ORF931; ORF938; ORF939; ORF958; ORF959; ORF960; ORF966; ORF995; ORF1021; ORF1040; ORF1041; ORF1042; ORF1085; ORF1100; ORF1102; ORF1117; ORF1118; ORF1119; ORF1120; ORF1135 and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the intermediate metabolism of nucleotides or nucleic acids, such as for example CTP synthetase or GMP synthetase, and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF77; ORF78; ORF138; ORF189; ORF190; ORF233; ORF246; ORF338; ORF412; ORF421; ORF438; ORF607; ORF648; ORF657; ORF740; ORF783; ORF967; ORF989; ORF990; ORF992; ORF1011; ORF1058; ORF1059; ORF1073; ORF1074 and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the metabolism of nucleic acids, such as for example DNA polymerases or DNA topoisomerases, and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF14; ORF59; ORF70; ORF71; ORF97; ORF113; ORF137; ORF141; ORF169; ORF285; ORF287; ORF288; ORF313; ORF326; ORF358; ORF411; ORF443; ORF548; ORF569; ORF601; ORF651; ORF654; ORF658; ORF659; ORF664; ORF665; ORF694; ORF698; ORF704; ORF760; ORF762; ORF763; ORF786; ORF787; ORF788; ORF801; ORF802; ORF812; ORF819; ORF822; ORF870; ORF897; ORF898; ORF902; ORF908; ORF916; ORF954; ORF955; ORF961; ORF983; ORF996; ORF1007; ORF1012; ORF1013; ORF1014; ORF1015; ORF1038; ORF1137 and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the metabolism of amino acids or polypeptides, such as for example serine hydroxymethyl transferase or the proteins which load amino acids onto transfer RNAs, and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF99; ORF111; ORF127; ORF134; ORF140; ORF174; ORF175; ORF176; ORF353; ORF377; ORF404; ORF523; ORF539; ORF559; ORF561; ORF586; ORF598; ORF609; ORF636; ORF687; ORF700; ORF701; ORF759; ORF790; ORF857; ORF861; ORF904; ORF936; ORF952; ORF962; ORF963; ORF964; ORF965; ORF991; ORF1003; ORF1004; ORF1005; ORF1018; ORF1067; ORF1110; ORF1111; ORF1112; ORF1114; ORF1121; ORF1122; ORF1123; ORF1124; ORF1125 and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the metabolism of polypeptides, such as for example protein kinases or proteases, and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF4; ORF44; ORF45; ORF48; ORF54; ORF112; ORF130; ORF155; ORF163; ORF212; ORF257; ORF307; ORF343; ORF405; ORF416; ORF458; ORF540; ORF541; ORF542; ORF543; ORF544; ORF560; ORF594; ORF652; ORF699; ORF723; ORF747; ORF817; ORF827; ORF871; ORF909; ORF910; ORF911; ORF912; ORF1023; ORF1051; ORF1052; ORF1081 and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the metabolism of fatty acids, such as for example succinyl-CoA-synthesizing proteins or phosphatidylserine synthetase, and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF76; ORF284; ORF308; ORF309; ORF310; ORF311; ORF312; ORF425; ORF433; ORF565; ORF688; ORF690; ORF691; ORF767; ORF797; ORF894; ORF895; ORF994; ORF1020; ORF1030; ORF1033; ORF1034; ORF1046; ORF1047; ORF1057 and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the synthesis of the wall, such as for example KDO transferase, and the proteins responsible for the attachment of certain sugars onto the exposed proteins, and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF49; ORF50; ORF177; ORF178; ORF245; ORF610; ORF972; ORF974; ORF978; ORF1037 and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the transcription, translation and/or maturation process, such as for example initiation factors, RNA polymerases or certain chaperone proteins, and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF90; ORF92; ORF131; ORF151; ORF199; ORF333; ORF334; ORF336; ORF379; ORF589; ORF590; ORF619; ORF630; ORF649; ORF739; ORF741; ORF806; ORF821; ORF843; ORF968; ORF971; ORF1061 and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae ribosomal polypeptide or one of its representative fragments, such as for example the ribosomal proteins L21, L27 and S10, and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF93; ORF94; ORF95; ORF136; ORF259; ORF332; ORF348; ORF583; ORF584; ORF588; ORF591; ORF592; ORF663; ORF666; ORF667; ORF669; ORF670; ORF671; ORF672; ORF673; ORF674; ORF675; ORF676; ORF677; ORF678; ORF679; ORF680; ORF681; ORF683; ORF684; ORF738; ORF781; ORF1008; ORF1024; ORF1025; ORF1066 and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae transport polypeptide or one of its representative fragments, such as for example the proteins for transporting amino acids, sugars and certain oligopeptides, and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF40; ORF41; ORF52; ORF105; ORF106; ORF107; ORF109; ORF133; ORF210; ORF211; ORF214; ORF215; ORF216; ORF217; ORF218; ORF219; ORF220; ORF223; ORF242; ORF260; ORF293; ORF299; ORF366; ORF369; ORF575; ORF602; ORF638; ORF639; ORF640; ORF643; ORF653; ORF702; ORF703; ORF724; ORF732; ORF855; ORF856; ORF901; ORF906; ORF933; ORF942; ORF1043; ORF1086; ORF1105 and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the virulence process, such as for example the proteins analogous to the Escherichia coli vacB protein, and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF546; ORF550; ORF778; ORF779; ORF886 and one of their representative fragments.

Preferably, the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the secretory system and/or which is secreted, such as for example proteins homologous to proteins in the secretory system of certain bacteria such as the Salmonellae or the Yersiniae, and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF751; ORF874; ORF875; ORF876; ORF883; ORF884; ORF885 and one of their representative fragments.

Preferably, the invention also relates to a nucleotide sequence according to the invention, characterized in that they encode a polypeptide specific to Chlamydia pneumoniae or one of its representative fragments (with a Blast E value of >10⁻⁵), and in that they comprise a nucleotide sequence chosen from the following sequences:

ORF7; ORF8; ORF17; ORF18; ORF19; ORF20; ORF22; ORF23; ORF24; ORF51; ORF60; ORF63; ORF65; ORF66; ORF67; ORF83; ORF84; ORF86; ORF87; ORF125; ORF143; ORF144; ORF179; ORF182; ORF184; ORF185; ORF187; ORF221; ORF252; ORF254;; ORF278; ORF279; ORF387; ORF388; ORF397; ORF1048; ORF1049; ORF1050; ORF1128; ORF1130; ORF1131 and one of their representative fragments.

Also forming part of the invention are polypeptides encoded by the polynucleotides of the invention, as well as fusion polypeptides comprising such polypeptides. In one embodiment, the polypeptides and fusion polypeptides immunoreact with seropositive serum of an individual infected with Chlamydia pneumoniae. For example, described below, are polypeptide sequences exhibiting particularly preferable characteristics. For each group of preferred polypeptides described below, it is to be understood that in addition to the individual polypeptides listed, in instances wherein such polypeptides are encoded as part of “combined” ORFs, such “combined” polypeptides are also to be included within the preferred group.

The subject of the invention is also a polypeptide according to the invention, characterized in that it is a polypeptide of the cellular envelope, preferably of the outer cellular envelope, of Chlamydia pneumoniae or one of its representative fragments. According to the invention, the said polypeptide is preferably chosen from the polypeptides having the following sequences:

SEQ ID No. 15; SEQ ID No. 25; SEQ ID No. 26; SEQ ID No. 27; SEQ ID No. 28; SEQ ID No. 29; SEQ ID No. 30; SEQ ID No. 31; SEQ ID No. 32; SEQ ID No. 33; SEQ ID No. 35; SEQ ID No. 68; SEQ ID No. 124; SEQ ID No. 275; SEQ ID No. 291; SEQ ID No. 294; SEQ ID No. 327; SEQ ID No. 342; SEQ ID No. 364; SEQ ID No. 374; SEQ ID No. 380; SEQ ID No. 414; SEQ ID No. 439; SEQ ID No. 466; SEQ ID No. 467; SEQ ID No. 468; SEQ ID No. 469; SEQ ID No. 470; SEQ ID No. 472; SEQ ID No. 474; SEQ ID No. 476; SEQ ID No. 477; SEQ ID No. 478; SEQ ID No. 479; SEQ ID No. 480; SEQ ID No. 482; SEQ ID No. 485; SEQ ID No. 500; SEQ ID No. 501; SEQ ID No. 503; SEQ ID No. 504; SEQ ID No. 505; SEQ ID No. 506; SEQ ID No. 520; SEQ ID No. 578; SEQ ID No. 580; SEQ ID No. 581; SEQ ID No. 595; SEQ ID No. 596; SEQ ID No. 597; SEQ ID No. 737; SEQ ID No. 830; SEQ ID No. 834; SEQ ID No. 836; SEQ ID No. 893; SEQ ID No. 917; SEQ ID No. 932; SEQ ID No. 976; SEQ ID No. 1035; SEQ ID No. 1045; SEQ ID No. 1090 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae transmembrane polypeptide or one of its representative fragments, having between 1 and 3 transmembrane domains, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 2; SEQ ID No. 3; SEQ ID No. 6; SEQ ID No. 9; SEQ ID No. 10; SEQ ID No. 11; SEQ ID No. 13; SEQ ID No. 14; SEQ ID No. 16; SEQ ID No. 18; SEQ ID No. 19; SEQ ID No. 20; SEQ ID No. 21; SEQ ID No. 22; SEQ ID No. 25; SEQ ID No. 27; SEQ ID No. 28; SEQ ID No. 29; SEQ ID No. 30; SEQ ID No. 31; SEQ ID No. 32; SEQ ID No. 33; SEQ ID No. 34; SEQ ID No. 35; SEQ ID No. 37; SEQ ID No. 39; SEQ ID No. 41; SEQ ID No. 42; SEQ ID No. 44; SEQ ID No. 45; SEQ ID No. 46; SEQ ID No. 47; SEQ ID No. 48; SEQ ID No. 49; SEQ ID No. 50; SEQ ID No. 53; SEQ ID No. 54; SEQ ID No. 56; SEQ ID No. 57; SEQ ID No. 59; SEQ ID No. 60; SEQ ID No. 61; SEQ ID No. 62; SEQ ID No. 63; SEQ ID No. 64; SEQ ID No. 65; SEQ ID No. 66; SEQ ID No. 69;; SEQ ID No. 72; SEQ ID No. 73; SEQ ID No. 74; SEQ ID No. 76; SEQ ID No. 77; SEQ ID No. 78; SEQ ID No. 79; SEQ ID No. 80; SEQ ID No. 82; SEQ ID No. 84; SEQ ID No. 85; SEQ ID No. 86; SEQ ID No. 88; SEQ ID No. 89; SEQ ID No. 90; SEQ ID No. 91; SEQ ID No. 92; SEQ ID No. 93; SEQ ID No. 95; SEQ ID No. 96; SEQ ID No. 98; SEQ ID No. 99; SEQ ID No. 100; SEQ ID No. 101; SEQ ID No. 102; SEQ ID No. 103; SEQ ID No. 104; SEQ ID No. 105; SEQ ID No. 106; SEQ ID No. 107; SEQ ID No. 108; SEQ ID No. 114; SEQ ID No. 117; SEQ ID No. 118; SEQ ID No. 122; SEQ ID No. 123; SEQ ID No. 124; SEQ ID No. 125; SEQ ID No. 129; SEQ ID No. 130; SEQ ID No. 131; SEQ ID No. 132; SEQ ID No. 133; SEQ ID No. 134; SEQ ID No. 135; SEQ ID No. 137; SEQ ID No. 138; SEQ ID No. 139; SEQ ID No. 140; SEQ ID No. 141; SEQ ID No. 142; SEQ ID No. 143; SEQ ID No. 145; SEQ ID No. 146; SEQ ID No. 147; SEQ ID No. 150; SEQ ID No. 151; SEQ ID No. 152; SEQ ID No. 156; SEQ ID No. 157; SEQ ID No. 158; SEQ ID No. 159; SEQ ID No. 160; SEQ ID No. 161; SEQ ID No. 162; SEQ ID No. 164; SEQ ID No. 166; SEQ ID No. 167; SEQ ID No. 170; SEQ ID No. 173; SEQ ID No. 175; SEQ ID No. 176; SEQ ID No. 178; SEQ ID No. 179; SEQ ID No. 180; SEQ ID No. 182; SEQ ID No. 183; SEQ ID No. 184; SEQ ID No. 185; SEQ ID No. 186; SEQ ID No. 187; SEQ ID No. 188; SEQ ID No. 189; SEQ ID No. 190; SEQ ID No. 191; SEQ ID No. 192; SEQ ID No. 194; SEQ ID No. 195; SEQ ID No. 196; SEQ ID No. 197; SEQ ID No. 198; SEQ ID No. 199; SEQ ID No. 200; SEQ ID No. 201; SEQ ID No. 202; SEQ ID No. 205; SEQ ID No. 207; SEQ ID No. 208; SEQ ID No. 209; SEQ ID No. 210; SEQ ID No. 212; SEQ ID No. 215; SEQ ID No. 219; SEQ ID No. 220; SEQ ID No. 224; SEQ ID No. 226; SEQ ID No. 227; SEQ ID No. 228; SEQ ID No. 231; SEQ ID No. 232; SEQ ID No. 233; SEQ ID No. 234; SEQ ID No. 235; SEQ ID No. 236; SEQ ID No. 238; SEQ ID No. 239; SEQ ID No. 240; SEQ ID No. 241; SEQ ID No. 242; SEQ ID No. 244; SEQ ID No. 247; SEQ ID No. 251; SEQ ID No. 252; SEQ ID No. 253; SEQ ID No. 255; SEQ ID No. 256; SEQ ID No. 257; SEQ ID No. 258; SEQ ID No. 260; SEQ ID No. 262; SEQ ID No. 263; SEQ ID No. 266; SEQ ID No. 267; SEQ ID No. 268; SEQ ID No. 269; SEQ ID No. 270; SEQ ID No. 273; SEQ ID No. 274; SEQ ID No. 276; SEQ ID No. 278; SEQ ID No. 279; SEQ ID No. 280; SEQ ID No. 281; SEQ ID No. 282; SEQ ID No. 283; SEQ ID No. 284; SEQ ID No. 286; SEQ ID No. 287; SEQ ID No. 289; SEQ ID No. 290; SEQ ID No. 291; SEQ ID No. 293; SEQ ID No. 294; SEQ ID No. 297; SEQ ID No. 304; SEQ ID No. 305; SEQ ID No. 307; SEQ ID No. 308; SEQ ID No. 309; SEQ ID No. 310; SEQ ID No. 311; SEQ ID No. 313; SEQ ID No. 314; SEQ ID No. 315; SEQ ID No. 316; SEQ ID No. 318; SEQ ID No. 319; SEQ ID No. 320; SEQ ID No. 321; SEQ ID No. 322; SEQ ID No. 323; SEQ ID No. 324; SEQ ID No. 325; SEQ ID No. 326; SEQ ID No. 331; SEQ ID No. 332; SEQ ID No. 336; SEQ ID No. 338; SEQ ID No. 339; SEQ ID No. 341; SEQ ID No. 344; SEQ ID No. 345; SEQ ID No. 346; SEQ ID No. 350; SEQ ID No. 352; SEQ ID No. 353; SEQ ID No. 356; SEQ ID No. 357; SEQ ID No. 358; SEQ ID No. 359; SEQ ID No. 360; SEQ ID No. 362; SEQ ID No. 365; SEQ ID No. 366; SEQ ID No. 367; SEQ ID No. 370; SEQ ID No. 372; SEQ ID No. 373; SEQ ID No. 376; SEQ ID No. 377; SEQ ID No. 378; SEQ ID No. 379; SEQ ID No. 381; SEQ ID No. 382; SEQ ID No. 383; SEQ ID No. 384; SEQ ID No. 385; SEQ ID No. 386; SEQ ID No. 387; SEQ ID No. 390; SEQ ID No. 392; SEQ ID No. 393; SEQ ID No. 394; SEQ ID No. 396; SEQ ID No. 398; SEQ ID No. 399; SEQ ID No. 400; SEQ ID No. 404; SEQ ID No. 408; SEQ ID No. 410; SEQ ID No. 411; SEQ ID No. 413; SEQ ID No. 4 16; SEQ ID No. 41 7; SEQ ID No. 41 8; SEQ ID No. 420; SEQ ID No. 422; SEQ ID No. 424; SEQ ID No. 427; SEQ ID No. 428; SEQ ID No. 429; SEQ ID No. 430; SEQ ID No. 431; SEQ ID No. 433; SEQ ID No. 434; SEQ ID No. 437; SEQ ID No. 440; SEQ ID No. 441; SEQ ID No. 442; SEQ ID No. 443; SEQ ID No. 444; SEQ ID No. 445; SEQ ID No. 447; SEQ ID No. 450; SEQ ID No. 451; SEQ ID No. 452; SEQ ID No. 455; SEQ ID No. 456; SEQ ID No. 459; SEQ ID No. 460; SEQ ID No. 461; SEQ ID No. 462; SEQ ID No. 463; SEQ ID No. 464; SEQ ID No. 465; SEQ ID No. 467; SEQ ID No. 469; SEQ ID No. 471; SEQ ID No. 474; SEQ ID No. 475; SEQ ID No. 476; SEQ ID No. 477; SEQ ID No. 479; SEQ ID No. 482; SEQ ID No. 483; SEQ ID No. 484; SEQ ID No. 485; SEQ ID No. 486; SEQ ID No. 487; SEQ ID No. 488; SEQ ID No. 491; SEQ ID No. 493; SEQ ID No. 494; SEQ ID No. 497; SEQ ID No. 498; SEQ ID No. 499; SEQ ID No. 503; SEQ ID No. 508; SEQ ID No. 509; SEQ ID No. 510; SEQ ID No. 512; SEQ ID No. 514; SEQ ID No. 515; SEQ ID No. 516; SEQ ID No. 517; SEQ ID No. 518; SEQ ID No. 520; SEQ ID No. 521; SEQ ID No. 523; SEQ ID No. 525; SEQ ID No. 527; SEQ ID No. 528; SEQ ID No. 529; SEQ ID No. 530; SEQ ID No. 531; SEQ ID No. 533; SEQ ID No. 534; SEQ ID No. 535; SEQ ID No. 536; SEQ ID No. 537; SEQ ID No. 540; SEQ ID No. 541; SEQ ID No. 543; SEQ ID No. 544; SEQ ID No. 545; SEQ ID No. 546; SEQ ID No. 548; SEQ ID No. 549; SEQ ID No. 551; SEQ ID No. 553; SEQ ID No. 554; SEQ ID No. 555; SEQ ID No. 556; SEQ ID No. 557; SEQ ID No. 558; SEQ ID No. 559; SEQ ID No. 560; SEQ ID No. 562; SEQ ID No. 563; SEQ ID No. 564; SEQ ID No. 565; SEQ ID No. 566; SEQ ID No. 569; SEQ ID No. 571; SEQ ID No. 573; SEQ ID No. 576; SEQ ID No. 577; SEQ ID No. 581; SEQ ID No. 583; SEQ ID No. 584; SEQ ID No. 585; SEQ ID No. 586; SEQ ID No. 588; SEQ ID No. 591; SEQ ID No. 592; SEQ ID No. 594; SEQ ID No. 595; SEQ ID No. 596; SEQ ID No. 597; SEQ ID No. 599; SEQ ID No. 600; SEQ ID No. 603; SEQ ID No. 605; SEQ ID No. 608; SEQ ID No. 614; SEQ ID No. 615; SEQ ID No. 620; SEQ ID No. 621; SEQ ID No. 622; SEQ ID No. 623; SEQ ID No. 624; SEQ ID No. 625; SEQ ID No. 629; SEQ ID No. 630; SEQ ID No. 631; SEQ ID No. 633; SEQ ID No. 634; SEQ ID No. 637; SEQ ID No. 642; SEQ ID No. 644; SEQ ID No. 645; SEQ ID No. 647; SEQ ID No. 648; SEQ ID No. 652; SEQ ID No. 654; SEQ ID No. 655; SEQ ID No. 657; SEQ ID No. 658; SEQ ID No. 659; SEQ ID No. 660; SEQ ID No. 661; SEQ ID No. 664; SEQ ID No. 665; SEQ ID No. 666; SEQ ID No. 667; SEQ ID No. 670; SEQ ID No. 671; SEQ ID No. 672; SEQ ID No. 673; SEQ ID No. 674; SEQ ID No. 676; SEQ ID No. 679; SEQ ID No. 681; SEQ ID No. 684; SEQ ID No. 687; SEQ ID No. 688; SEQ ID No. 689; SEQ ID No. 690; SEQ ID No. 693; SEQ ID No. 694; SEQ ID No. 695; SEQ ID No. 696; SEQ ID No. 697; SEQ ID No. 698; SEQ ID No. 699; SEQ ID No. 700; SEQ ID No. 701; SEQ ID No. 703; SEQ ID No. 705; SEQ ID No. 706; SEQ ID No. 707; SEQ ID No. 708; SEQ ID No. 710; SEQ ID No. 712; SEQ ID No. 715; SEQ ID No. 716; SEQ ID No. 717; SEQ ID No. 718; SEQ ID No. 719; SEQ ID No. 721; SEQ ID No. 722; SEQ ID No. 723; SEQ ID No. 725; SEQ ID No. 726; SEQ ID No. 727; SEQ ID No. 728; SEQ ID No. 729; SEQ ID No. 730; SEQ ID No. 731; SEQ ID No. 733; SEQ ID No. 736; SEQ ID No. 737; SEQ ID No. 738; SEQ ID No. 740; SEQ ID No. 741; SEQ ID No. 742; SEQ ID No. 743; SEQ ID No. 747; SEQ ID No. 748; SEQ ID No. 750; SEQ ID No. 752; SEQ ID No. 754; SEQ ID No. 755; SEQ ID No. 756; SEQ ID No. 757; SEQ ID No. 759; SEQ ID No. 760; SEQ ID No. 761; SEQ ID No. 762; SEQ ID No. 763; SEQ ID No. 764; SEQ ID No. 765; SEQ ID No. 766; SEQ ID No. 767; SEQ ID No. 768; SEQ ID No. 772; SEQ ID No. 774; SEQ ID No. 775; SEQ ID No. 777; SEQ ID No. 781; SEQ ID No. 783; SEQ ID No. 788; SEQ ID No. 791; SEQ ID No. 792; SEQ ID No. 793; SEQ ID No. 794; SEQ ID No. 795; SEQ ID No. 796; SEQ ID No. 797; SEQ ID No. 798; SEQ ID No. 799; SEQ ID No. 802; SEQ ID No. 803; SEQ ID No. 806; SEQ ID No. 807; SEQ ID No. 808; SEQ ID No. 809; SEQ ID No. 810; SEQ ID No. 811; SEQ ID No. 813; SEQ ID No. 814; SEQ ID No. 815; SEQ ID No. 816; SEQ ID No. 817; SEQ ID No. 819; SEQ ID No. 820; SEQ ID No. 821; SEQ ID No. 823; SEQ ID No. 824; SEQ ID No. 827; SEQ ID No. 829; SEQ ID No. 830; SEQ ID No. 831; SEQ ID No. 833; SEQ ID No. 834; SEQ ID No. 835; SEQ ID No. 837; SEQ ID No. 844; SEQ ID No. 845 ; SEQ ID No. 8 46; SEQ ID No. 8 47 ; SEQ ID No. 848; SEQ ID No. 849; SEQ ID No. 850; SEQ ID No. 851; SEQ ID No. 852; SEQ ID No. 854; SEQ ID No. 855; SEQ ID No. 856; SEQ ID No. 857; SEQ ID No. 859; SEQ ID No. 860; SEQ ID No. 862; SEQ ID No. 865; SEQ ID No. 866; SEQ ID No. 868; SEQ ID No. 869; SEQ ID No. 870; SEQ ID No. 871; SEQ ID No. 872; SEQ ID No. 874; SEQ ID No. 877; SEQ ID No. 878; SEQ ID No. 879; SEQ ID No. 880; SEQ ID No. 881; SEQ ID No. 882; SEQ ID No. 884; SEQ ID No. 885; SEQ ID No. 888; SEQ ID No. 889; SEQ ID No. 890; SEQ ID No. 891; SEQ ID No. 892; SEQ ID No. 894; SEQ ID No. 895; SEQ ID No. 896; SEQ ID No. 897; SEQ ID No. 899; SEQ ID No. 900; SEQ ID No. 902; SEQ ID No. 903; SEQ ID No. 904; SEQ ID No. 905; SEQ ID No. 909; SEQ ID No. 910; SEQ ID No. 912; SEQ ID No. 913; SEQ ID No. 914; SEQ ID No. 915; SEQ ID No. 917; SEQ ID No. 918; SEQ ID No. 919; SEQ ID No. 921; SEQ ID No. 923; SEQ ID No. 924; SEQ ID No. 926; SEQ ID No. 927; SEQ ID No. 928; SEQ ID No. 929; SEQ ID No. 930; SEQ ID No. 931; SEQ ID No. 937; SEQ ID No. 938; SEQ ID No. 939; SEQ ID No. 941; SEQ ID No. 943; SEQ ID No. 948; SEQ ID No. 951; SEQ ID No. 952; SEQ ID No. 953; SEQ ID No. 958; SEQ ID No. 960; SEQ ID No. 963; SEQ ID No. 964; SEQ ID No. 965; SEQ ID No. 968; SEQ ID No. 970; SEQ ID No. 974; SEQ ID No. 975; SEQ ID No. 977; SEQ ID No. 979; SEQ ID No. 980; SEQ ID No. 981; SEQ ID No. 983; SEQ ID No. 984; SEQ ID No. 985; SEQ ID No. 987; SEQ ID No. 989; SEQ ID No. 992; SEQ ID No. 993; SEQ ID No. 997; SEQ ID No. 998; SEQ ID No. 999; SEQ ID No. 1001; SEQ ID No. 1002; SEQ ID No. 1004; SEQ ID No. 1005; SEQ ID No. 1009; SEQ ID No. 1013; SEQ ID No. 1014; SEQ ID No. 1015; SEQ ID No. 1016; SEQ ID No. 1019; SEQ ID No. 1021; SEQ ID No. 1023; SEQ ID No. 1024; SEQ ID No. 1029; SEQ ID No. 1031; SEQ ID No. 1033; SEQ ID No. 1034; SEQ ID No. 1039; SEQ ID No. 1041; SEQ ID No. 1042; SEQ ID No. 1045; SEQ ID No. 1047; SEQ ID No. 1049; SEQ ID No. 1051; SEQ ID No. 1052; SEQ ID No. 1053; SEQ ID No. 1054; SEQ ID No. 1056; SEQ ID No. 1059; SEQ ID No. 1061; SEQ ID No. 1062; SEQ ID No. 1063; SEQ ID No. 1064; SEQ ID No. 1065; SEQ ID No. 1067; SEQ ID No. 1075; SEQ ID No. 1077; SEQ ID No. 1078; SEQ ID No. 1079; SEQ ID No. 1080; SEQ ID No. 1081; SEQ ID No. 1089; SEQ ID No. 1095; SEQ ID No. 1097; SEQ ID No. 1098; SEQ ID No. 1099; SEQ ID No. 1101; SEQ ID No. 1102; SEQ ID No. 1103; SEQ ID No. 1106; SEQ ID No. 1107; SEQ ID No. 1108; SEQ ID No. 1109; SEQ ID No. 1110; SEQ ID No. 1113; SEQ ID No. 1116; SEQ ID No. 1118; SEQ ID No. 1119; SEQ ID No. 1121; SEQ ID No. 1123; SEQ ID No. 1124; SEQ ID No. 1126; SEQ ID No. 1128; SEQ ID No. 1130; SEQ ID No. 1131; SEQ ID No. 1133; SEQ ID No. 1134; SEQ ID No. 1136; SEQ ID No. 1137 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae transmembrane polypeptide or one of its representative fragments, having between 4 and 6 transmembrane domains, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 5; SEQ ID No. 7; SEQ ID No. 8; SEQ ID No. 15; SEQ ID No. 36; SEQ ID No. 38; SEQ ID No. 51; SEQ ID No. 55; SEQ ID No. 58; SEQ ID No. 67; SEQ ID No. 70; SEQ ID No. 81; SEQ ID No. 97; SEQ ID No. 110; SEQ ID No. 111; SEQ ID No. 115; SEQ ID No. 119; SEQ ID No. 126; SEQ ID No. 128; SEQ ID No. 148; SEQ ID No. 155; SEQ ID No. 163; SEQ ID No. 165; SEQ ID No. 168; SEQ ID No. 169; SEQ ID No. 171; SEQ ID No. 172; SEQ ID No. 174; SEQ ID No. 177; SEQ ID No. 181; SEQ ID No. 193; SEQ ID No. 203; SEQ ID No. 213; SEQ ID No. 214; SEQ ID No. 216; SEQ ID No. 217; SEQ ID No. 221; SEQ ID No. 222; SEQ ID No. 225; SEQ ID No. 229; SEQ ID No. 243; SEQ ID No. 246; SEQ ID No. 248; SEQ ID No. 254; SEQ ID No. 261; SEQ ID No. 285; SEQ ID No. 288; SEQ ID No. 292; SEQ ID No. 296; SEQ ID No. 298; SEQ ID No. 299; SEQ ID No. 301; SEQ ID No. 303; SEQ ID No. 317; SEQ ID No. 328; SEQ ID No. 329; SEQ ID No. 351; SEQ ID No. 354; SEQ ID No. 355; SEQ ID No. 364; SEQ ID No. 371; SEQ ID No. 374; SEQ ID No. 375; SEQ ID No. 391; SEQ ID No. 395; SEQ ID No. 401; SEQ ID No. 403; SEQ ID No. 405; SEQ ID No. 409; SEQ ID No. 414; SEQ ID No. 419; SEQ ID No. 421; SEQ ID No. 423; SEQ ID No. 425; SEQ ID No. 438; SEQ ID No. 448; SEQ ID No. 453; SEQ ID No. 458; SEQ ID No. 466; SEQ ID No. 468; SEQ ID No. 470; SEQ ID No. 480; SEQ ID No. 489; SEQ ID No. 490; SEQ ID No. 496; SEQ ID No. 501; SEQ ID No. 504; SEQ ID No. 505; SEQ ID No. 506; SEQ ID No. 511; SEQ ID No. 513; SEQ ID No. 519; SEQ ID No. 526; SEQ ID No. 532; SEQ ID No. 538; SEQ ID No. 539; SEQ ID No. 547; SEQ ID No. 550; SEQ ID No. 561; SEQ ID No. 568; SEQ ID No. 570; SEQ ID No. 574; SEQ ID No. 578; SEQ ID No. 579; SEQ ID No. 580; SEQ ID No. 582; SEQ ID No. 589; SEQ ID No. 593; SEQ ID No. 598; SEQ ID No. 601; SEQ ID No. 604; SEQ ID No. 610; SEQ ID No. 613; SEQ ID No. 617; SEQ ID No. 626; SEQ ID No. 632; SEQ ID No. 635; SEQ ID No. 638; SEQ ID No. 640; SEQ ID No. 641; SEQ ID No. 646; SEQ ID No. 649; SEQ ID No. 650; SEQ ID No. 651; SEQ ID No. 686; SEQ ID No. 711; SEQ ID No. 724; SEQ ID No. 732; SEQ ID No. 734; SEQ ID No. 744; SEQ ID No. 745; SEQ ID No. 749; SEQ ID No. 751; SEQ ID No. 769; SEQ ID No. 770; SEQ ID No. 771; SEQ ID No. 773; SEQ ID No. 776; SEQ ID No. 779; SEQ ID No. 780; SEQ ID No. 785; SEQ ID No. 787; SEQ ID No. 789; SEQ ID No. 801; SEQ ID No. 805; SEQ ID No. 812; SEQ ID No. 822; SEQ ID No. 825; SEQ ID No. 826; SEQ ID No. 839; SEQ ID No. 841; SEQ ID No. 843; SEQ ID No. 853; SEQ ID No. 861; SEQ ID No. 875; SEQ ID No. 876; SEQ ID No. 886; SEQ ID No. 893; SEQ ID No. 898; SEQ ID No. 906; SEQ ID No. 907; SEQ ID No. 908; SEQ ID No. 920; SEQ ID No. 922; SEQ ID No. 925; SEQ ID No. 933; SEQ ID No. 935; SEQ ID No. 936; SEQ ID No. 944; SEQ ID No. 946; SEQ ID No. 947; SEQ ID No. 954; SEQ ID No. 959; SEQ ID No. 961; SEQ ID No. 966; SEQ ID No. 967; SEQ ID No. 972; SEQ ID No. 978; SEQ ID No. 995; SEQ ID No. 996; SEQ ID No. 1000; SEQ ID No. 1003; SEQ ID No. 1010; SEQ ID No. 1011; SEQ ID No. 1012; SEQ ID No. 1017; SEQ ID No. 1020; SEQ ID No. 1030; SEQ ID No. 1036; SEQ ID No. 1038; SEQ ID No. 1043; SEQ ID No. 1046; SEQ ID No. 1048; SEQ ID No. 1050; SEQ ID No. 1058; SEQ ID No. 1071; SEQ ID No. 1073; SEQ ID No. 1084; SEQ ID No. 1085; SEQ ID No. 1086; SEQ ID No. 1087; SEQ ID No. 1091; SEQ ID No. 1092; SEQ ID No. 1094; SEQ ID No. 1096; SEQ ID No. 1100; SEQ ID No. 1104; SEQ ID No. 1111; SEQ ID No. 1112; SEQ ID No. 1114; SEQ ID No. 1117; SEQ ID No. 1122; SEQ ID No. 1125 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae transmembrane polypeptide or one of its representative fragments, having at least 7 transmembrane domains, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 17; SEQ ID No. 52; SEQ ID No. 68; SEQ ID No. 83; SEQ ID No. 87; SEQ ID No. 109 ; SEQ ID No. 112; SEQ ID No. 113; SEQ ID No. 120; SEQ ID No. 121; SEQ ID No. 127; SEQ ID No. 153; SEQ ID No. 204; SEQ ID No. 211; SEQ ID No. 218; SEQ ID No. 223; SEQ ID No. 275; SEQ ID No. 277; SEQ ID No. 295; SEQ ID No. 300; SEQ ID No. 302; SEQ ID No. 306; SEQ ID No. 327; SEQ ID No. 335; SEQ ID No. 342; SEQ ID No. 343; SEQ ID No. 347; SEQ ID No. 349; SEQ ID No. 361; SEQ ID No. 363; SEQ ID No. 369; SEQ ID No. 380; SEQ ID No. 388; SEQ ID No. 389; SEQ ID No. 397; SEQ ID No. 415; SEQ ID No. 432; SEQ ID No. 439; SEQ ID No. 446; SEQ ID No. 449; SEQ ID No. 472; SEQ ID No. 478; SEQ ID No. 500; SEQ ID No. 522; SEQ ID No. 524; SEQ ID No. 567; SEQ ID No. 575; SEQ ID No. 602; SEQ ID No. 606; SEQ ID No. 609; SEQ ID No. 636; SEQ ID No. 639; SEQ ID No. 643; SEQ ID No. 653; SEQ ID No. 668; SEQ ID No. 692; SEQ ID No. 702; SEQ ID No. 704; SEQ ID No. 713; SEQ ID No. 720; SEQ ID No. 778; SEQ ID No. 784; SEQ ID No. 800; SEQ ID No. 836; SEQ ID No. 838; SEQ ID No. 842; SEQ ID No. 864; SEQ ID No. 867; SEQ ID No. 883; SEQ ID No. 901; SEQ ID No. 916; SEQ ID No. 932; SEQ ID No. 934; SEQ ID No. 940; SEQ ID No. 942; SEQ ID No. 950; SEQ ID No. 956; SEQ ID No. 971; SEQ ID No. 973; SEQ ID No. 976; SEQ ID No. 988; SEQ ID No. 994; SEQ ID No. 1018; SEQ ID No. 1028; SEQ ID No. 1035; SEQ ID No. 1037; SEQ ID No. 1044; SEQ ID No. 1055; SEQ ID No. 1057; SEQ ID No. 1068; SEQ ID No. 1069; SEQ ID No. 1070; SEQ ID No. 1072; SEQ ID No. 1082; SEQ ID No. 1088; SEQ ID No. 1105; SEQ ID No. 1132; SEQ ID No. 1135 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, in that it is a Chlamydia pneumoniae surface exposed polypeptide or one of its representative fragments, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 15, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 27, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 36, SEQ ID No. 1257, SEQ ID No. 280, SEQ ID No. 291, SEQ ID No. 314, SEQ ID No. 354, SEQ ID No. 380, SEQ ID No. 1266, SEQ ID No. 466, SEQ ID No. 467, SEQ ID No. 468, SEQ ID No. 469, SEQ ID No. 470, SEQ ID No. 472, SEQ ID No. 474, SEQ ID No. 476, SEQ ID No. 477, SEQ ID No. 478, SEQ ID No. 479, SEQ ID No. 480, SEQ ID No. 482, SEQ ID No. 483, SEQ ID No. 485, SEQ ID No. 486, SEQ ID No. 500, SEQ ID No. 501, SEQ ID No. 503, SEQ ID No. 504, SEQ ID No. 505, SEQ ID No. 506, SEQ ID No. 507, SEQ ID No. 1268, SEQ ID No. 1269, SEQ ID No. 543, SEQ ID No. 544, SEQ ID No. 578, SEQ ID No. 579, SEQ ID No. 580, SEQ ID No. 581, SEQ ID No. 595, SEQ ID No. 596, SEQ ID No. 597, SEQ ID No. 1271, SEQ ID No. 633, SEQ ID No. 637, SEQ ID No. 699, SEQ ID No. 706, SEQ ID No. 737, SEQ ID No. 744, SEQ ID No. 1273, SEQ ID No. 751, SEQ ID No. 775, SEQ ID No. 776, SEQ ID No. 777, SEQ ID No. 793, SEQ ID No. 815, SEQ ID No. 830, SEQ ID No. 1221, SEQ ID No. 849, SEQ ID No. 851, SEQ ID No. 852, SEQ ID No. 874, SEQ ID No. 891, SEQ ID No. 922, SEQ ID No. 940, SEQ ID No. 1231, SEQ ID No. 1281, SEQ ID No. 1035, SEQ ID No. 1079, SEQ ID No. 1087, SEQ ID No. 1108, and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae lipoprotein or one of its representative fragments, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 3, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 16, SEQ ID No. 1254, SEQ ID No. 1255, SEQ ID No. 38, SEQ ID No. 1256, SEQ ID No. 62, SEQ ID No. 85, SEQ ID No. 1258, SEQ ID No. 115, SEQ ID No. 1151, SEQ ID No. 151, SEQ ID No. 1259, SEQ ID No. 173, SEQ ID No. 1261, SEQ ID No. 186, SEQ ID No. 194, SEQ ID No. 205, SEQ ID No. 214, SEQ ID No. 216, SEQ ID No. 217, SEQ ID No. 238, SEQ ID No. 1177, SEQ ID No. 280, SEQ ID No. 291, SEQ ID No. 317, SEQ ID No. 327, SEQ ID No. 354, SEQ ID No. 364, SEQ ID No. 367, SEQ ID No. 414, SEQ ID No. 432, SEQ ID No. 1192, SEQ ID No. 460, SEQ ID No. 1267, SEQ ID No. 1268, SEQ ID No. 520, SEQ ID No. 536, SEQ ID No. 1270, SEQ ID No. 576, SEQ ID No. 597, SEQ ID No. 603, SEQ ID No. 609, SEQ ID No. 637, SEQ ID No. 1272, SEQ ID No. 652, SEQ ID No. 1213, SEQ ID No. 699, SEQ ID No. 705, SEQ ID No. 706, SEQ ID No. 708, SEQ ID No. 711, SEQ ID No. 727, SEQ ID No. 1274, SEQ ID No. 800, SEQ ID No. 814, SEQ ID No. 825, SEQ ID No. 829, SEQ ID No. 830, SEQ ID No. 831, SEQ ID No. 844, SEQ ID No. 849, SEQ ID No. 1275, SEQ ID No. 1276, SEQ ID No. 1277, SEQ ID No. 872, SEQ ID No. 878, SEQ ID No. 880, SEQ ID No. 891, SEQ ID No. 892, SEQ ID No. 1278, SEQ ID No. 1279, SEQ ID No. 1280, SEQ ID No. 941, SEQ ID No. 942, SEQ ID No. 1282, SEQ ID No. 1283, SEQ ID No. 952, SEQ ID No. 988, SEQ ID No. 998, SEQ ID No. 1009, SEQ ID No. 1285, SEQ ID No. 1235, SEQ ID No. 1028, SEQ ID No. 1056, SEQ ID No. 1070, SEQ ID No. 1287, SEQ ID No. 1087, SEQ ID No. 1288, SEQ ID No. 1289, SEQ ID No. 1098, SEQ ID No. 1246, SEQ ID No. 1291, SEQ ID No. 1108, SEQ ID No. 1109, SEQ ID No. 1112, SEQ ID No. 1133, and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, in that it is a Chlamydia pneucmoniae polypeptide involved in lipopolysaccharide (LPS) biosynthesis, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 316, SEQ ID No. 564, SEQ ID No. 610, SEQ ID No. 647, SEQ ID No. 1211, SEQ ID No. 688, SEQ ID No. 924, and one of their representative fragments.

Preferably, the invention relates to additional LPS-related polypeptides according to the invention, in that it is:

(a) a Chlamydia pneumoniae KDO (3-deoxy-D-manno-octylosonic acid)-related polypeptide or one of its representative fragments, and in that it is chosen from the polypeptides having the following sequences: SEQ ID No. 177, SEQ ID No. 1156, SEQ ID No. 245, SEQ ID No. 767, and one of their representative fragments;

(b) a Chlamydia pneumoniae phosphomannomutase-related polypeptide or one of its representative fragments, and in that it is chosen from the polypeptides having the following sequences: SEQ ID No. 74, and its representative fragment;

(c) a Chlamydia pneumoniae phosphoglucomutase-related polypeptide or one of its representative fragments, and in that it is chosen from the polypeptides having the following sequences: SEQ ID No. 1286, SEQ ID No. 1039, and its representative fragment; and

(d) a Chlamydia pneumoniae lipid A component-related polypeptide or one of its representative fragments, and in that it is chosen from the polypeptides having the following sequences: SEQ ID No. 689, SEQ ID No. 690, SEQ ID No. 691, SEQ ID No. 1037, and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments that contains an RGD sequence and is also an outer membrane protein, and in that it is chosen from the polypeptides having the following sequences: SEQ ID No. 468 and its representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments that contains an RGD sequence that shows homology to cds1, cds2, and copN type III virulence loci in Chlamydia Psitacci, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 350 and its representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments that is cysteine-rich and contains RGD sequence, and in that it is chosen from the polypeptides having the following sequences: SEQ ID No. 1290, SEQ ID No. 6846, SEQ ID No. 6848, and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, in that it is a Chlamydia pneumoniae outer membrane polypeptide that contains cysteines in their first 30 amino acids and also contain an RGD sequence, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 114, SEQ ID No. 170, SEQ ID No. 171, SEQ ID No. 1264, SEQ ID No. 268, SEQ ID No. 1265, SEQ ID No. 350, SEQ ID No. 393, SEQ ID No. 394, SEQ ID No. 451, SEQ ID No. 452, SEQ ID No. 453, SEQ ID No. 473, SEQ ID No. 499, SEQ ID No. 515, SEQ ID No. 519, SEQ ID No. 525, SEQ ID No. 526, SEQ ID No. 538, SEQ ID No. 611, SEQ ID No. 645, SEQ ID No. 686, SEQ ID No. 700, SEQ ID No. 746, SEQ ID No. 755, SEQ ID No. 756, SEQ ID No. 757, SEQ ID No. 789, SEQ ID No. 814, SEQ ID No. 855, SEQ ID No. 856, SEQ ID No. 878, SEQ ID No. 957, SEQ ID No. 958, SEQ ID No. 989, SEQ ID No. 1290, and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments that contains RGD sequences homologous to Chlamydia trachomatis polypeptides containing RGD sequences, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 114, SEQ ID No. 468, SEQ ID No. 755, SEQ ID No. 756, SEQ ID No. 757, SEQ ID No. 855, SEQ ID No. 856, SEQ ID No. 905, SEQ ID No. 913, SEQ ID No. 914, SEQ ID No. 915, and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, in that it is a Chlamydia pneumoniae Type III and non-Type III secreted polypeptide or one of its representative fragments, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 25, SEQ ID No. 28, SEQ ID No. 29, SEQ ID No. 33, SEQ ID No. 308, SEQ ID No. 309, SEQ ID No. 343, SEQ ID No. 344, SEQ ID No. 345, SEQ ID No. 367, SEQ ID No. 414, SEQ ID No. 415, SEQ ID No. 480, SEQ ID No. 550, SEQ ID No. 579, SEQ ID No. 580, SEQ ID No. 581, SEQ ID No. 597, SEQ ID No. 699, SEQ ID No. 744, SEQ ID No. 751, SEQ ID No. 776, SEQ ID No. 866, SEQ ID No. 874, SEQ ID No. 883, SEQ ID No. 884, SEQ ID No. 888, SEQ ID No. 891, SEQ ID No. 6845, and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, in that it is a Chlamydia pneumoniae cell wall anchored surface polypeptide or one of its representative fragments, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 267, SEQ ID No. 271, SEQ ID No. 419, SEQ ID No. 590, SEQ ID No. 932, SEQ ID No. 6844, SEQ ID No. 6847, and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments not found in Chlamydia trachomatis (Blastp P>e⁻¹⁰), and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 1254, SEQ ID No. 23, SEQ ID No. 1255, SEQ ID No. 24, SEQ ID No. 1139, SEQ ID No. 1140, SEQ ID No. 46, SEQ ID No. 47, SEQ ID No. 51, SEQ ID No. 60, SEQ ID No. 1256, SEQ ID No. 61, SEQ ID No. 62, SEQ ID No. 63, SEQ ID No. 64, SEQ ID No. 1257, SEQ ID No. 65, SEQ ID No. 66, SEQ ID No. 67, SEQ ID No. 68, SEQ ID No. 1143, SEQ ID No. 1145, SEQ ID No. 83, SEQ ID No. 84, SEQ ID No. 1146, SEQ ID No. 85, SEQ ID No. 86, SEQ ID No. 87, SEQ ID No. 1258, SEQ ID No. 116, SEQ ID No. 117, SEQ ID No. 125, SEQ ID No. 1148, SEQ ID No. 143, SEQ ID No. 1150, SEQ ID No. 1151, SEQ ID No. 144, SEQ ID No. 145, SEQ ID No. 147, SEQ ID No. 148, SEQ ID No. 149, SEQ ID No. 150, SEQ ID No. 152, SEQ ID No. 1259, SEQ ID No. 162, SEQ ID No. 166, SEQ ID No. 1154, SEQ ID No. 167, SEQ ID No. 1261, SEQ ID No. 1156, SEQ ID No. 1157, SEQ ID No. 178, SEQ ID No. 179, SEQ ID No. 1158, SEQ ID No. 182, SEQ ID No. 183, SEQ ID No. 184, SEQ ID No. 185, SEQ ID No. 1159, SEQ ID No. 186, SEQ ID No. 1160, SEQ ID No. 187, SEQ ID No. 188, SEQ ID No. 189, SEQ ID No. 190, SEQ ID No. 1161, SEQ ID No. 1162, SEQ ID No. 191, SEQ ID No. 192, SEQ ID No. 194, SEQ ID No. 195, SEQ ID No. 1163, SEQ ID No. 196, SEQ ID No. 201, SEQ ID No. 202, SEQ ID No. 209, SEQ ID No. 212, SEQ ID No. 221, SEQ ID No. 224, SEQ ID No. 1167, SEQ ID No. 226, SEQ ID No. 227, SEQ ID No. 228, SEQ ID No. 229, SEQ ID No. 230, SEQ ID No. 231, SEQ ID No. 232, SEQ ID No. 1169, SEQ ID No. 1170, SEQ ID No. 1171, SEQ ID No. 234, SEQ ID No. 235, SEQ ID No. 236, SEQ ID No. 1172, SEQ ID No. 243, SEQ ID No. 251, SEQ ID No. 252, SEQ ID No. 1176, SEQ ID No. 253, SEQ ID No. 255, SEQ ID No. 254, SEQ ID No. 256, SEQ ID No. 1177, SEQ ID No. 1178, SEQ ID No. 262, SEQ ID No. 263, SEQ ID No. 1264, SEQ ID No. 278, SEQ ID No. 279, SEQ ID No. 1180, SEQ ID No. 280, SEQ ID No. 290, SEQ ID No. 291, SEQ ID No. 292, SEQ ID No. 296, SEQ ID No. 1181, SEQ ID No. 297, SEQ ID No. 298, SEQ ID No. 300, SEQ ID No. 1265, SEQ ID No. 322, SEQ ID No. 324, SEQ ID No. 325, SEQ ID No. 370, SEQ ID No. 1186, SEQ ID No. 371, SEQ ID No. 372, SEQ ID No. 1187, SEQ ID No. 373, SEQ ID No. 378, SEQ ID No. 1266, SEQ ID No. 382, SEQ ID No. 383, SEQ ID No. 384, SEQ ID No. 385, SEQ ID No. 386, SEQ ID No. 1188, SEQ ID No. 1189, SEQ ID No. 391, SEQ ID No. 392, SEQ ID No. 398, SEQ ID No. 400, SEQ ID No. 403, SEQ ID No. 1191, SEQ ID No. 423, SEQ ID No. 435, SEQ ID No. 445, SEQ ID No. 450, SEQ ID No. 1193, SEQ ID No. 456, SEQ ID No. 460, SEQ ID No. 461, SEQ ID No. 465, SEQ ID No. 1196, SEQ ID No. 471, SEQ ID No. 473, SEQ ID No. 475, SEQ ID No. 481, SEQ ID No. 484, SEQ ID No. 487, SEQ ID No. 488, SEQ ID No. 489, SEQ ID No. 490, SEQ ID No. 491, SEQ ID No. 492, SEQ ID No. 493, SEQ ID No. 494, SEQ ID No. 495, SEQ ID No. 496, SEQ ID No. 497, SEQ ID No. 498, SEQ ID No. 499, SEQ ID No. 502, SEQ ID No. 1267, SEQ ID No. 1268, SEQ ID No. 508, SEQ ID No. 510, SEQ ID No. 509, SEQ ID No. 512, SEQ ID No. 515, SEQ ID No. 519, SEQ ID No. 1197, SEQ ID No. 521, SEQ ID No. 1198, SEQ ID No. 522, SEQ ID No. 524, SEQ ID No. 528, SEQ ID No. 534, SEQ ID No. 537, SEQ ID No. 1269, SEQ ID No. 1270, SEQ ID No. 548, SEQ ID No. 551, SEQ ID No. 557, SEQ ID No. 1201, SEQ ID No. 1203, SEQ ID No. 562, SEQ ID No. 566, SEQ ID No. 593, SEQ ID No. 595, SEQ ID No. 600, SEQ ID No. 1271, SEQ ID No. 604, SEQ ID No. 611, SEQ ID No. 612 , SEQ ID No. 614, SEQ ID No. 616, SEQ ID No. 625, SEQ ID No. 627, SEQ ID No. 628, SEQ ID No. 629, SEQ ID No. 631, SEQ ID No. 641, SEQ ID No. 1272, SEQ ID No. 648, SEQ ID No. 1212, SEQ ID No. 663, SEQ ID No. 685, SEQ ID No. 707, SEQ ID No. 714, SEQ ID No. 715, SEQ ID No. 716, SEQ ID No. 717, SEQ ID No. 722, SEQ ID No. 746, SEQ ID No. 1273, SEQ ID No. 761, SEQ ID No. 764, SEQ ID No. 770, SEQ ID No. 1217, SEQ ID No. 783, SEQ ID No. 1274, SEQ ID No. 803, SEQ ID No. 815, SEQ ID No. 1220, SEQ ID No. 835, SEQ ID No. 1221, SEQ ID No. 844, SEQ ID No. 845, SEQ ID No. 846, SEQ ID No. 847, SEQ ID No. 848, SEQ ID No. 849, SEQ ID No. 850, SEQ ID No. 851, SEQ ID No. 1275, SEQ ID No. 852, SEQ ID No. 862, SEQ ID No. 1276, SEQ ID No. 1277, SEQ ID No. 873, SEQ ID No. 1223, SEQ ID No. 892, SEQ ID No. 919, SEQ ID No. 1225, SEQ ID No. 1278, SEQ ID No. 926, SEQ ID No. 1228, SEQ ID No. 1229, SEQ ID No. 1230, SEQ ID No. 1279, SEQ ID No. 1281, SEQ ID No. 1282, SEQ ID No. 1283, SEQ ID No. 948, SEQ ID No. 950, SEQ ID No. 949, SEQ ID No. 951, SEQ ID No. 980, SEQ ID No. 982, SEQ ID No. 1233, SEQ ID No. 999, SEQ ID No. 1000, SEQ ID No. 1001, SEQ ID No. 1002, SEQ ID No. 1008, SEQ ID No. 1285, SEQ ID No. 1235, SEQ ID No. 1016, SEQ ID No. 1019, SEQ ID No. 1027, SEQ ID No. 1036, SEQ ID No. 1241, SEQ ID No. 1048, SEQ ID No. 1049, SEQ ID No. 1050, SEQ ID No. 1053, SEQ ID No. 1054, SEQ ID No. 1064, SEQ ID No. 1076, SEQ ID No. 1091, SEQ ID No. 1288, SEQ ID No. 1093, SEQ ID No. 1289, SEQ ID No. 1101, SEQ ID No. 1103, SEQ ID No. 1245, SEQ ID No. 1246, SEQ ID No. 1247, SEQ ID No. 1290, SEQ ID No. 1291, SEQ ID No. 1115, SEQ ID No. 1116, SEQ ID No. 1118, SEQ ID No. 1120, SEQ ID No. 1249, SEQ ID No. 1121, SEQ ID No. 1250, SEQ ID No. 1126, SEQ ID No. 1251, SEQ ID No. 1127, SEQ ID No. 1128, SEQ ID No. 1130, SEQ ID No. 1129, SEQ ID No. 1131, SEQ ID No. 1136, SEQ ID No. 1253, SEQ ID No. 6844, SEQ ID No. 6846, SEQ ID No. 6847, SEQ ID No. 6848, and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the intermediate metabolism, in particular in the metabolism of sugars and/or of cofactors, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 2; SEQ ID No. 55; SEQ ID No. 56; SEQ ID No. 69; SEQ ID No. 75; SEQ ID No. 80; SEQ ID No. 100; SEQ ID No. 110; SEQ ID No. 114; SEQ ID No. 120; SEQ ID No. 121; SEQ ID No. 157; SEQ ID No. 160; SEQ ID No. 161; SEQ ID No. 172; SEQ ID No. 180; SEQ ID No. 181; SEQ ID No. 198; SEQ ID No. 200; SEQ ID No. 225; SEQ ID No. 248; SEQ ID No. 249; SEQ ID No. 276; SEQ ID No. 277; SEQ ID No. 318; SEQ ID No. 319; SEQ ID No. 320; SEQ ID No. 323; SEQ ID No. 331; SEQ ID No. 347; SEQ ID No. 375; SEQ ID No. 376; SEQ ID No. 381; SEQ ID No. 393; SEQ ID No. 394; SEQ ID No. 395; SEQ ID No. 396; SEQ ID No. 409; SEQ ID No. 446; SEQ ID No. 447; SEQ ID No. 448; SEQ ID No. 449; SEQ ID No. 513; SEQ ID No. 516; SEQ ID No. 571; SEQ ID No. 647; SEQ ID No. 662; SEQ ID No. 697; SEQ ID No. 718; SEQ ID No. 793; SEQ ID No. 794; SEQ ID No. 808; SEQ ID No. 809; SEQ ID No. 838; SEQ ID No. 839; SEQ ID No. 840; SEQ ID No. 853; SEQ ID No. 854; SEQ ID No. 918; SEQ ID No. 923; SEQ ID No. 929; SEQ ID No. 931; SEQ ID No. 938; SEQ ID No. 939; SEQ ID No. 958; SEQ ID No. 959; SEQ ID No. 960; SEQ ID No. 966; SEQ ID No. 995; SEQ ID No. 1021; SEQ ID No. 1040; SEQ ID No. 1041; SEQ ID No. 1042; SEQ ID No. 1085; SEQ ID No. 1100; SEQ ID No. 1 102; SEQ ID No. 1117; SEQ ID No. 1118; SEQ ID No. 1119; SEQ ID No. 1120; SEQ ID No. 1135 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the intermediate metabolism of nucleotides or nucleic acids, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 77; SEQ ID No. 78; SEQ ID No. 138; SEQ ID No. 189; SEQ ID No. 190; SEQ ID No. 233; SEQ ID No. 246; SEQ ID No. 338; SEQ ID No. 412; SEQ ID No. 421; SEQ ID No. 438; SEQ ID No. 607; SEQ ID No. 648; SEQ ID No. 657; SEQ ID No. 740; SEQ ID No. 783; SEQ ID No. 967; SEQ ID No. 989; SEQ ID No. 990; SEQ ID No. 992; SEQ ID No. 1011; SEQ ID No. 1058; SEQ ID No. 1059; SEQ ID No. 1073; SEQ ID No. 1074 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the metabolism of nucleic acids, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 14; SEQ ID No. 59; SEQ ID No. 70; SEQ ID No. 71; SEQ ID No. 97; SEQ ID No. 113; SEQ ID No. 137; SEQ ID No. 141; SEQ ID No. 169; SEQ ID No. 285; SEQ ID No. 287; SEQ ID No. 288; SEQ ID No. 313; SEQ ID No. 326; SEQ ID No. 358; SEQ ID No. 411; SEQ ID No. 443; SEQ ID No. 548; SEQ ID No. 569; SEQ ID No. 601; SEQ ID No. 651; SEQ ID No. 654; SEQ ID No. 658; SEQ ID No. 659; SEQ ID No. 664; SEQ ID No. 665; SEQ ID No. 694; SEQ ID No. 698; SEQ ID No. 704; SEQ ID No. 760; SEQ ID No. 762; SEQ ID No. 763; SEQ ID No. 786; SEQ ID No. 787; SEQ ID No. 788; SEQ ID No. 801; SEQ ID No. 802; SEQ ID No. 812; SEQ ID No. 819; SEQ ID No. 822; SEQ ID No. 870; SEQ ID No. 897; SEQ ID No. 898; SEQ ID No. 902; SEQ ID No. 908; SEQ ID No. 916; SEQ ID No. 954; SEQ ID No. 955; SEQ ID No. 961; SEQ ID No. 983; SEQ ID No. 996; SEQ ID No. 1007; SEQ ID No. 1012; SEQ ID No. 1013; SEQ ID No. 1014; SEQ ID No. 1015; SEQ ID No. 1038; SEQ ID No. 1137 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the metabolism of amino acids or polypeptides, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 99; SEQ ID No. 111; SEQ ID No. 127; SEQ ID No. 134; SEQ ID No. 140; SEQ ID No. 174; SEQ ID No. 175; SEQ ID No. 176; SEQ ID No. 353; SEQ ID No. 377; SEQ ID No. 404; SEQ ID No. 523; SEQ ID No. 539; SEQ ID No. 559; SEQ ID No. 561; SEQ ID No. 586; SEQ ID No. 598; SEQ ID No. 609; SEQ ID No. 636; SEQ ID No. 687; SEQ ID No. 700; SEQ ID No. 701; SEQ ID No. 759; SEQ ID No. 790; SEQ ID No. 857; SEQ ID No. 861; SEQ ID No. 904; SEQ ID No. 936; SEQ ID No. 952; SEQ ID No. 962; SEQ ID No. 963; SEQ ID No. 964; SEQ ID No. 965; SEQ ID No. 991; SEQ ID No. 1003; SEQ ID No. 1004; SEQ ID No. 1005; SEQ ID No. 1018; SEQ ID No. 1067; SEQ ID No. 1110; SEQ ID No. 1111; SEQ ID No. 1112; SEQ ID No. 1114; SEQ ID No. 1121; SEQ ID No. 1122; SEQ ID No. 1123; SEQ ID No. 1124; SEQ ID No. 1125 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the metabolism of polypeptides, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 4; SEQ ID No. 44; SEQ ID No. 45; SEQ ID No. 48; SEQ ID No. 54; SEQ ID No. 112; SEQ ID No. 130; SEQ ID No. 155; SEQ ID No. 163; SEQ ID No. 212; SEQ ID No. 257; SEQ ID No. 307; SEQ ID No. 343; SEQ ID No. 405; SEQ ID No. 416; SEQ ID No. 458; SEQ ID No. 540; SEQ ID No. 541; SEQ ID No. 542; SEQ ID No. 543; SEQ ID No. 544; SEQ ID No. 560; SEQ ID No. 594; SEQ ID No. 652; SEQ ID No. 699; SEQ ID No. 723; SEQ ID No. 747; SEQ ID No. 817; SEQ ID No. 827; SEQ ID No. 871; SEQ ID No. 909; SEQ ID No. 910; SEQ ID No. 911; SEQ ID No. 912; SEQ ID No. 1023; SEQ ID No. 1051; SEQ ID No. 1052; SEQ ID No. 1081 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the metabolism of fatty acids, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 76; SEQ ID No. 284; SEQ ID No. 308; SEQ ID No. 309; SEQ ID No. 310; SEQ ID No. 311; SEQ ID No. 312; SEQ ID No. 425; SEQ ID No. 433; SEQ ID No. 565; SEQ ID No. 688; SEQ ID No. 690; SEQ ID No. 691; SEQ ID No. 767; SEQ ID No. 797; SEQ ID No. 894; SEQ ID No. 895; SEQ ID No. 994; SEQ ID No. 1020; SEQ ID No. 1030; SEQ ID No. 1033; SEQ ID No. 1034; SEQ ID No. 1046; SEQ ID No. 1047; SEQ ID No. 1057 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the synthesis of the wall, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 49; SEQ ID No. 50; SEQ ID No. 177; SEQ ID No. 178; SEQ ID No. 245; SEQ ID No. 610; SEQ ID No. 972; SEQ ID No. 974; SEQ ID No. 978; SEQ ID No. 1037 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the transcription, translation and/or maturation process, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 90; SEQ ID No. 92; SEQ ID No. 131; SEQ ID No. 151; SEQ ID No. 199; SEQ ID No. 333; SEQ ID No. 334; SEQ ID No. 336; SEQ ID No. 379; SEQ ID No. 589; SEQ ID No. 590; SEQ ID No. 619; SEQ ID No. 630; SEQ ID No. 649; SEQ ID No. 739; SEQ ID No. 741; SEQ ID No. 806; SEQ ID No. 821; SEQ ID No. 843; SEQ ID No. 968; SEQ ID No. 971; SEQ ID No. 1061 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae ribosomal polypeptide or one of its representative fragments, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 93; SEQ ID No. 94; SEQ ID No. 95; SEQ ID No. 136; SEQ ID No. 259; SEQ ID No. 332; SEQ ID No. 348; SEQ ID No. 583; SEQ ID No. 584; SEQ ID No. 588; SEQ ID No. 591; SEQ ID No. 592; SEQ ID No. 663; SEQ ID No. 666; SEQ ID No. 667; SEQ ID No. 669; SEQ ID No. 670; SEQ ID No. 671; SEQ ID No. 672; SEQ ID No. 673; SEQ ID No. 674; SEQ ID No. 675; SEQ ID No. 676; SEQ ID No. 677; SEQ ID No. 678; SEQ ID No. 679; SEQ ID No. 680; SEQ ID No. 681; SEQ ID No. 683; SEQ ID No. 684; SEQ ID No. 738; SEQ ID No. 781; SEQ ID No. 1008; SEQ ID No. 1024; SEQ ID No. 1025; SEQ ID No. 1066 and one of their representative fragments.

Preferably, the invention also relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae transport polypeptide or one of its representative fragments, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 40; SEQ ID No. 41; SEQ ID No. 52; SEQ ID No. 105; SEQ ID No. 106; SEQ ID No. 107; SEQ ID No. 109; SEQ ID No. 133; SEQ ID No. 210; SEQ ID No. 211; SEQ ID No. 214; SEQ ID No. 215; SEQ ID No. 216; SEQ ID No. 217; SEQ ID No. 218; SEQ ID No. 219; SEQ ID No. 220; SEQ ID No. 223; SEQ ID No. 242; SEQ ID No. 260; SEQ ID No. 293; SEQ ID No. 299; SEQ ID No. 366; SEQ ID No. 369; SEQ ID No. 575; SEQ ID No. 602; SEQ ID No. 638; SEQ ID No. 639; SEQ ID No. 640; SEQ ID No. 643; SEQ ID No. 653; SEQ ID No. 702; SEQ ID No. 703; SEQ ID No. 724; SEQ ID No. 732; SEQ ID No. 855; SEQ ID No. 856; SEQ ID No. 901; SEQ ID No. 906; SEQ ID No. 933; SEQ ID No. 942; SEQ ID No. 1043; SEQ ID No. 1086; SEQ ID No. 1105 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the virulence process, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 546; SEQ ID No. 550; SEQ ID No. 778; SEQ ID No. 779; SEQ ID No. 886 and one of their representative fragments.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a Chlamydia pneumoniae polypeptide or one of its representative fragments which is involved in the secretory system and/or which is secreted, and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 751; SEQ ID No. 874; SEQ ID No. 875; SEQ ID No. 876; SEQ ID No. 883; SEQ ID No. 884; SEQ ID No. 885 and one of their representative fragments.

The secreted polypeptides, including the Type III and other, non-Type III secreted polypeptides, of the present invention, as well as the corresponding nucleotide sequences, may be detected by techniques known to persons skilled in the art, such as for example the techniques using cloning combined with vectors allowing the expression of the said polypeptides fused to export markers such as the luc gene for luciferase or the PhoA gene for alkaline phosphatase.

Preferably, the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide specific to Chlamydia pneumoniae or one of its representative fragments(with a Blast E value of >10⁻⁵), and in that it is chosen from the polypeptides having the following sequences:

SEQ ID No. 7; SEQ ID No. 8; SEQ ID No. 17; SEQ ID No. 18; SEQ ID No. 19; SEQ ID No. 20; SEQ ID No. 22; SEQ ID No. 23; SEQ ID No. 24; SEQ ID No. 51; SEQ ID No. 60; SEQ ID No. 63; SEQ ID No. 65; SEQ ID No. 66; SEQ ID No. 67; SEQ ID No. 83; SEQ ID No. 84; SEQ ID No. 86; SEQ ID No. 87; SEQ ID No. 125; SEQ ID No. 143; SEQ ID No. 144; SEQ ID No. 179; SEQ ID No. 182; SEQ ID No. 184; SEQ ID No. 185; SEQ ID No. 187; SEQ ID No. 221; SEQ ID No. 252; SEQ ID No. 254;; SEQ ID No. 278; SEQ ID No. 279; SEQ ID No. 387; SEQ ID No. 388; SEQ ID No. 397; SEQ ID No. 1048; SEQ ID No. 1049; SEQ ID No. 1050; SEQ ID No. 1128; SEQ ID No. 1130; SEQ ID No. 1131 and one of their representative fragments.

In general, in the present invention, the functional group to which a polypeptide of the invention belongs, as well as its corresponding nucleotide sequence, may be determined either by comparative analogy with sequences already known, or by the use of standard techniques of biochemistry, of cytology combined with the techniques of genetic engineering such as immunoaffinity, localization by immunolabelling, differential extraction, measurement of enzymatic activity, study of the activity inducing or repressing expression or the study of expression in E. coli.

It is clearly understood, on the one hand, that, in the present invention, the nucleotide sequences (ORF) and the amino acid sequences (SEQ ID No. 2 to SEQ ID No. 1291 and SEQ ID No. 6844 to SEQ ID No. 684) which are listed by functional group, are not exhaustive within the group considered. Moreover, it is also clearly understood that, in the present invention, a nucleotide sequence (ORF) or an amino acid sequence mentioned within a given functional group may also be part of another group taking into account, for example, the interrelationship between the groups listed. Accordingly, and as an example of this interrelationship, an exported and/or secreted polypeptide as well as its coding nucleotide sequence may also be involved in the Chlamydia pneumoniae virulence process by modifying the defense mechanism of the infected host cell, or a transmembrane polypeptide or its coding nucleotide sequence is also part of the polypeptides or coding nucleotide sequences of the cellular envelope.

The subject of the present invention is also the nucleotide and/or polypeptide sequences according to the invention, characterized in that the said sequences are recorded on a medium, called recording medium, whose type and nature facilitate the reading, the analysis and the exploitation of the said sequences. These media may of course also contain other information extracted from the present invention, such as in particular the analogies with already known sequences, such as those mentioned in Table 1 of the present description, and/or may contain, in addition, information relating to the nucleotide and/or polypeptide sequences of other microorganisms so as to facilitate the comparative analysis and the exploitation of the results obtained.

Among these recording media, computer-readable media, such as magnetic, optical, electrical and hybrid media such as, for example, floppy disks, CD-ROMs or recording cassettes, are preferred in particular.

The invention also relates to nucleotide sequences which can be used as primer or probe, characterized in that the said sequences are chosen from the nucleotide sequences according to the invention.

The invention relates, in addition, to the use of a nucleotide sequence according to the invention, as primer or probe, for the detection and/or amplification of nucleic acid sequences.

The nucleotide sequences according to the invention may thus be used to amplify nucleotide sequences, in particular by the PCR technique (polymerase chain reaction) (Erlich, 1989; Innis et al., 1990; Rolfs et al., 1991, and White et al., 1997).

These oligodeoxyribonucleotide or oligoribonucleotide primers correspond to representative nucleotide fragments, and are advantageously at least 8 nucleotides, preferably at least 12 nucleotides, 15 nucleotides and still more preferably at least 20 nucleotides long.

Other techniques for amplifying the target nucleic acid may be advantageously used as alternatives to PCR.

The nucleotide sequences of the invention, in particular the primers according to the invention, may also be used in other methods for amplifying a target nucleic acid, such as:

the TAS (Transcription-based Amplification System) technique described by Kwoh et al. in 1989;

the 3SR (Self-Sustained Sequence Replication) technique described by Guatelli et al. in 1990;

the NASBA (Nucleic Acid Sequence Based Amplification) technique described by Kievitis et al. in 1991;

the SDA (Strand Displacement Amplification) technique (Walker et al., 1992);

the TMA (Transcription Mediated Amplification) technique.

The polynucleotides of the invention may also be used in techniques for amplifying or for modifying the nucleic acid serving as probe, such as:

the LCR (Ligase Chain Reaction) technique described by Landegren et al. in 1988 and perfected by Barany et al. in 1991, which uses a thermostable ligase;

the RCR (Repair Chain Reaction) technique described by Segev in 1992;

the CPR (Cycling Probe Reaction) technique described by Duck et al. in 1990;

the Q-beta-replicase amplification technique described by Miele et al. in 1983 and perfected in particular by Chu et al. in 1986, Lizardi et al. in 1988, and then by Burg et al. as well as by Stone et al. in 1996.

The invention also relates to the nucleotide sequences of fragments which can be obtained by amplification with the aid of at least one primer according to the invention. The present invention encompasses both hybridization probes and primers. In general, the complementary probes should be of a length sufficient to form a stable hybrid complex with the target sequences . Primers, while complementary to the target sequences need not form stable hybridization complexes with the target sequences alone. Rather, primers form stable complexes with the target sequences in the presence of polymerase to permit extension of the primer.

In the case where the target polynucleotide to be detected is possibly an RNA, for example an mRNA, it will be possible to use, prior to the use of an amplification reaction with the aid of at least one primer according to the invention or to the use of a method of detection with the aid of at least one probe of the invention, a reverse transcriptase-type enzyme so as to obtain a cDNA from the RNA contained in the biological sample. The cDNA obtained will then serve as target for the primer(s) or the probe(s) used in the amplification or detection method according to the invention.

The detection probe will be chosen so that it hybridizes with the target sequence or the amplicon generated from the target sequence. Such a detection probe will advantageously have as sequence a sequence of at least 12 nucleotides, in particular of at least 20 nucleotides, and preferably at least 100 nucleotides.

The invention also comprises the nucleotide sequences which can be used as probe or primer according to the invention, characterized in that they are labelled with a radioactive compound or with a nonradioactive compound.

The nonlabelled nucleotide sequences may be used directly as probes or primers; however, the sequences are generally labelled with a radioactive element (³²P, ³⁵S, ³H, ¹²⁵I) or with a nonradioactive molecule (biotin, acetylaminofluorene, digoxigenin, 5-bromo-deoxyuridine, fluorescein) so as to obtain probes which can be used in numerous applications.

Examples of nonradioactive labelling of nucleotide sequences are described, for example, in French patent No. 78,10975 or by Urdea et al. or by Sanchez-Pescador et al. in 1988.

In the latter case, one of the labelling methods described in patents FR-2 422 956 and FR-2 518 755 may also be used.

The invention also relates to the nucleotide sequences of fragments which can be obtained by hybridization with the aid of at least one probe according to the invention.

The hybridization technique may be performed in various ways (Matthews et al., 1988). The most common method consists in immobilizing the nucleic acid extracted from Chlamydia pneumoniae cells on a support (such as nitrocellulose, nylon, polystyrene) and in incubating, under well-defined conditions, the target nucleic acid immobilized with the probe. After hybridization, the excess probe is removed and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, of the fluorescence or of the enzymatic activity linked to the probe).

The invention also comprises the nucleotide sequences according to the invention, characterized in that they are covalently or noncovalently immobilized on a support.

According to another advantageous embodiment of the nucleic sequences according to the invention, the latter may be used immobilized on a support and may thus serve to capture, through specific hybridization, the target nucleic acid obtained from the biological sample to be tested. If necessary, the solid support is separated from the sample and the hybridization complex formed between the so-called capture probe and the target nucleic acid is then detected by means of a second probe, called detection probe, labelled with an easily detectable element.

The nucleotide sequences according to the invention may also be used in new analytical systems, DNA chips, which allow sequencing, the study of mutations and of the expression of genes, and which are currently of interest given their very small size and their high capacity in terms of number of analyses.

The principle of the operation of these chips is based on molecular probes, most often oligonucleotides, which are attached onto a miniaturized surface, generally of the order of a few square centimetres. During an analysis, a sample containing fragments of a target nucleic acid to be analysed, for example DNA or RNA labelled, for example, after amplification, is deposited onto the DNA chip in which the support has been coated beforehand with probes. Bringing the labelled target sequences into contact with the probes leads to the formation, through hybridization, of a duplex according to the rule of pairing defined by J. D. Watson and F. Crick. After a washing step, analysis of the surface of the chip allows the effective hybridizations to be located by means of the signals emitted by the labels tagging the target. A hybridization fingerprint results from this analysis which, by appropriate computer processing, will make it possible to determine information such as the presence of specific fragments in the sample, the determination of sequences and the presence of mutations.

The chip consists of a multitude of molecular probes, precisely organized or arrayed on a solid support whose surface is miniaturized. It is at the centre of a system where other elements (imaging system, microcomputer) allow the acquisition and interpretation of a hybridization fingerprint.

The hybridization supports are provided in the form of flat or porous surfaces (pierced with wells) composed of various materials. The choice of a support is determined by its physicochemical properties, or more precisely, by the relationship between the latter and the conditions under which the support will be placed during the synthesis or the attachment of the probes or during the use of the chip. It is therefore necessary, before considering the use of a particular support (R. S. Matson et al., 1994), to consider characteristics such as its stability to pH, its physical strength, its reactivity and its chemical stability as well as its capacity to nonspecifically bind nucleic acids. Materials such as glass, silicon and polymers are commonly used. Their surface is, in a first step, called “functionalization”, made reactive towards the groups which it is desired to attach thereon. After the functionalization, so-called spacer molecules are grafted onto the activated surface. Used as intermediates between the surface and the probe, these molecules of variable size render unimportant the surface properties of the supports, which often prove to be problematic for the synthesis or the attachment of the probes and for the hybridization.

Among the hybridization supports, there may be mentioned glass which is used, for example, in the method of in situ synthesis of oligonucleotides by photochemical addressing developed by the company Affymetrix (E. L. Sheldon, 1993), the glass surface being activated by silane. Genosensor Consortium (P. Mérel, 1994) also uses glass slides carrying wells 3 mm apart, this support being activated with epoxysilane.

Polymers or silicon may also be mentioned among these hybridization supports. For example, the Andrein Mirzabekov team has developed a chip consisting of polyacrylamide squares polymerized on a silanized glass surface (G. Yershov et al., 1996). Several teams use silicon, in particular the IFOS laboratory of Ecole Centrale of Lyon which uses a silicon semiconductor substrate which is p-doped by introducing it into its crystalline structure atoms whose valency is different from that of silicon. Various types of metals, in particular gold and platinum, may also be used as support (Genosensor Consortium (K. Beattie et al., 1993)).

The probes according to the invention may be synthesized directly in situ on the supports of the DNA chips. This in situ synthesis may be carried out by photochemical addressing (developed by the company Affymax (Amsterdam, Holland) and exploited industrially by its subsidiary Affymetrix (United States)) or based on the VLSIPS (very large scale immobilized polymer synthesis) technology (S. P. A. Fodor et al., 1991) which is based on a method of photochemically directed combinatory synthesis and the principle of which combines solid-phase chemistry, the use of photolabile protecting groups and photolithography.

The probes according to the invention may be attached to the DNA chips in various ways such as electrochemical addressing, automated addressing or the use of probe printers (T. Livache et al., 1994; G. Yershov et al., 1996; J. Derisi et al., 1996, and S. Borman, 1996).

The revealing of the hybridization between the probes of the invention, deposited or synthesized in situ on the supports of the DNA chips, and the sample to be analysed, may be determined, for example, by measurement of fluorescent signals, by radioactive counting or by electronic detection.

The use of fluorescent molecules such as fluorescein constitutes the most common method of labelling the samples. It allows direct or indirect revealing of the hybridization and allows the use of various fluorochromes.

Affymetrix currently provides an apparatus or a scanner designed to read its Gene Chip™ chips. It makes it possible to detect the hybridizations by scanning the surface of the chip in confocal microscopy (R. J. Lipshutz et al., 1995). Other methods of detecting fluorescent signals have been tested: coupling of an epifluorescence microscope and a CCD camera (G. Yershov et al., 1996), the use of an optical fibre collecting system (E. L. Sheldon, 1993). A conventional method consists in carrying out an end labelling, with phosphorus 32, of the target sequences, by means of an appropriate apparatus, the Phosphorimager (marketed by Molecular Dynamics). The electronic detection is based on the principle that the hybridization of two nucleic acid molecules is accompanied by physical phenomena which can be quantified under certain conditions (system developed by Ecole Centrale of Lyon and called GEN-FET (GEN field effect transistor)). Genosensor Consortium and the company Beckman Instruments who are developing an electronic chip or Permittivity Chips™ may also be mentioned (K. Beattie et al., 1993).

The nucleotide sequences according to the invention may thus be used in DNA chips to carry out the analysis of mutations. This analysis is based on the production of chips capable of analysing each base of a nucleotide sequence according to the invention.

The nucleotide sequences according to the invention may also be used in DNA chips to carry out the analysis of the expression of the Chlamydia pneumoniae genes. This analysis of the expression of Chlamydia pneumoniae genes is based on the use of chips where probes of the invention, chosen for their specificity to characterize a given gene, are present (D. J. Lockhart et al., 1996; D. D. Shoemaker et al., 1996). For the methods of analysis of gene expression using the DNA chips, reference may, for example, be made to the methods described by D. J. Lockhart et al. (1996) and Sosnowsky et al. (1997) for the synthesis of robes in situ or for the addressing and the attachment of previously synthesized probes. The target sequences to be analysed are labelled and in general fragmented into sequences of about 50 to 100 nucleotides before being hybridized onto the chip. After washing as described, for example, by D. J. Lockhart et al. (1996) and application of different electric fields (Sosnowsky et al., 1997), the labelled compounds are detected and quantified, the hybridizations being carried out at least in duplicate. Comparative analyses of the signal intensities obtained with respect to the same probe for different samples and/or for different probes with the same sample, determine the differential expression of RNA or of DNA derived from the sample.

The nucleotide sequences according to the invention may, in addition, be used in DNA chips where other nucleotide probes specific for other microorganisms are also present, and may allow the carrying out of a serial test allowing rapid identification of the presence of a microorganism in a sample.

Accordingly, the subject of the invention is also the nucleotide sequences according to the invention, characterized in that they are immobilized on a support of a DNA chip.

The DNA chips, characterized in that they contain at least one nucleotide sequence according to the invention, immobilized on the support of the said chip, also form part of the invention.

The said chips will preferably contain several probes or nucleotide sequences of the invention of different length and/or corresponding to different genes so as to identify, with greater certainty, the specificity of the target sequences or the desired mutation in the sample to be analysed.

Accordingly, the analyses carried out by means of primers and/or probes according to the invention, immobilized on supports such as DNA chips, will make it possible, for example, to identify, in samples, mutations linked to variations such as intraspecies variations. These variations may be correlated or associated with pathologies specific to the variant identified and will make it possible to select the appropriate treatment.

The invention thus comprises a DNA chip according to the invention, characterized in that it contains, in addition, at least one nucleotide sequence of a microorganism different from Chlamydia pneumoniae, immobilized on the support of the said chip; preferably, the different microorganism will be chosen from an associated microorganism, a bacterium of the Chlamydia family, and a variant of the species Chlamydia pneumoniae.

Another subject of the present invention is a vector for the cloning and/or the expression of a sequence, characterized in that it contains a nucleotide sequence according to the invention. Among the said vectors according to the invention, the vectors containing a nucleotide sequence encoding a polypeptide of the cellular, preferably outer, envelope of Chlamydia pneumoniae or one of its representative fragments, are preferred. In a specific embodiment, the vectors contain a nucleotide sequence encoding a Chlamydia pneumoniae secreted polypeptide or one of its representative fragments or encoding a transport polypeptide, a surface exposed polypeptide, a lipoprotein or one of its representative fragments, a polypeptide involved in lipopolysaccharide (LPS) biosynthesis, a Type III and non-Type III secreted polypeptide, a polypeptide containing RGD attachment sites, a cell wall anchored surface polypeptide, a polypeptide not found in Chlamydia trachomatis, a ribosomal polypeptide or a polypeptide involved in secretion, transcription, translation, maturation of proteins, a polypeptide involved in the synthesis of the wall, a polypeptide involved in the virulence, a polypeptide involved in the intermediate metabolism, in particular in the metabolism of sugars and/or of cofactors, a polypeptide involved in the metabolism of nucleotides, of amino acids, of nucleic acids or of fatty acids of Chlamydia pneumoniae or one of their representative fragments, or a polypeptide specific to Chlamydia pneumoniae.

According to the invention, the vectors comprise the elements necessary to allow the expression and/or the secretion of the said nucleotide sequences in a given host cell, and form part of the invention. The vector should, in this case, comprise a promoter, signals for initiation and for termination of translation, as well as appropriate regions for regulation of transcription. It should be capable of being stably maintained in the host cell and may optionally possess particular signals specifying the secretion of the translated protein. These different elements are chosen according to the host cell used. To this effect, the nucleotide sequences according to the invention may be inserted into autonomously-replicating vectors within the chosen host, or integrative vectors in the chosen host.

Any of the standard methods known to those skilled in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination).

Expression of a polypeptide, peptide or derivative, or analogs thereof encoded by a polynucleotide sequence in SEQ ID No. 1 or ORFs contained within SEQ ID No. 1 may be regulated by a second nucleic acid sequence so that the protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a protein or peptide may be controlled by any promoter/enhancer element known in the art. Promoters which may be used to control expression include, but are not limited to, the CMV promoter, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the $-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase promoter region (Herrera-Estrella et al., 1983, Nature 303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Gardner, et al., 1981, Nucl. Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).

The vectors according to the invention are, for example, vectors of plasmid or viral origin. In a specific embodiment, a vector is used that comprises a promoter operably linked to a protein or peptide-encoding a nucleic acid sequence in SEQ ID No. 1, or ORFs contained within SEQ ID No. 1, one or more origins of replication, and, optionally, one or more selectable markers (e.g., an antibiotic resistance gene). Expression vectors comprise regulatory sequences that control gene expression, including gene expression in a desired host cell. Preferred vectors for the expression of the polypeptides of the invention include the pET-type plasmid vectors (Promega) or pBAD plasmid vectors (Invitrogen). Furthermore, the vectors according to the invention are useful for transforming host cells so as to clone or express the nucleotide sequences of the invention.

Expression can also be achieved using targeted homologous recombination to activate Chlamydia pneumoniae genes present in the cloned genomic DNA. A heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous Chlamydia pneumoniae gene present in the cloned genome, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art (See, e.g., Chappel, U.S. Pat. No. 4,215,051 and Skoultchi, WO 91/06667 each of which is incorporated herein in its entirety).

Expression vector/host cell systems containing inserts of polynucleotide sequences in SEQ ID No. 1 or ORFs within SEQ ID No. 1, which encode polypeptides, peptides or derivatives, or analogs thereof, can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of “marker” gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a polynucleotide sequence inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted polynucleotide sequence. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain “marker” gene functions (e.g., thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of a polynucleotide sequence in the vector. For example, if the polynucleotide sequence in SEQ ID No. 1 or ORFs within SEQ ID No. 1 is inserted within the marker gene sequence of the vector, recombinants containing the insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the product of the polynucleotide sequence expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the expressed polypeptide in in vitro assay systems, e.g., binding with antibody, promotion of cell proliferation.

Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. The clones identified may be introduced into an appropriate host cell by standard methods, such as for example lipofection, electroporation, and heat shock. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity.

The invention also encompasses the host cells transformed by a vector according to the invention. These cells may be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, and then culturing the said cells under conditions allowing the replication and/or the expression of the transfected nucleotide sequence.

The host cell may be chosen from eukaryotic or prokaryotic systems, such as for example bacterial cells (Olins and Lee, 1993), but also yeast cells (Buckholz, 1993), as well as animal cells, in particular cultures of mammalian cells (Edwards and Aruffo, 1993), and in particular Chinese hamster ovary (CHO) cells, but also insect cells in which methods using baculoviruses for example may be used (Luckow, 1993).

Furthermore, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unglycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in mammalian cells can be used to ensure “native” glycosylation of a heterologous protein. Furthermore, different vector/host expression systems may effect processing reactions to different extents.

A preferred host cell for the expression of the proteins of the invention consists of prokaryotic cells, such as Gram bacteria. A further preferred host cell according to the invention is a bacterium belonging to the Chlamydia family, more preferably belonging to the species Chlamydia pneumoniae or chosen from a microorganism associated with the species Chlamydia pneumoniae.

In other specific embodiments, the polypeptides, peptides or derivatives, or analogs thereof may be expressed as a fusion, or chimeric protein product (comprising the protein, fragment, analog, or derivative joined via a peptide bond to a heterologous protein sequence (of a different protein)). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.

Genomic sequences can be cloned and expressed as translational gene products (i.e., peptides, polypeptides, and proteins) or transcriptional gene products (i.e., antisense and ribozymes).

The invention further relates to the intracellular production of an antisense nucleic acid sequence of SEQ ID No. 1 by transcription from an exogenous sequence. For example, a vector can be introduced in vivo such that it is taken up by a cell, within which cell the vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding an antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the an antisense RNA can be by any promoter known in the art to act in mammalian, preferably human, cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the CMV promoter, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3N long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42), etc.

In a specific embodiment, the antisense oligonucleotide comprises catalytic RNA, or a ribozyme (see, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225). In another embodiment, the oligonucleotide is a 2N-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analog (Inoue et al., 1987, FEBS Lett. 215:327-330).

In another embodiment, the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a polynucleotide sequence in SEQ ID No. 1. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acid sequence, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA transcribed from SEQ ID No. 1 may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

The invention also relates to the animals, except humans, comprising one of the above-described transformed cells according to the invention.

The production of transgenic animals according to the invention overexpressing one or more of the Chlamydia pneumoniae genes will be preferably carried out on rats, mice or rabbits according to methods well known to persons skilled in the art such as viral or nonviral transfections. The transgenic animals overexpressing one or more of the said genes may be obtained by transfection of multiple copies of the said genes under the control of a powerful promoter of a ubiquitous nature, or which is selective for one type of tissue. The transgenic animals may also be obtained by homologous recombination on embryonic stem cells, transfer of these stem cells to embryos, selection of the chimeras affected at the level of the reproductive lines, and growth of the said chimeras.

The transformed cells as well as the transgenic animals according to the invention can be used in methods of preparing the recombinant polypeptide.

It is now possible to produce recombinant polypeptides in a relatively large quantity by genetic engineering using the cells transformed with expression vectors according to the invention or using transgenic animals according to the invention.

The methods of preparing a polypeptide of the invention in recombinant form, characterized in that they use a vector and/or a cell transformed with a vector according to the invention and/or a transgenic animal comprising one of the said transformed cells according to the invention, are themselves included in the present invention.

Among the said methods of preparing a polypeptide of the invention in recombinant form, the methods of preparation using a vector, and/or a cell transformed with the said vector and/or a transgenic animal comprising one of the said transformed cells, containing a nucleotide sequence encoding a polypeptide of the cellular envelope of Chlamydia pneumoniae or one of its representative fragments, more preferably encoding a polypeptide of the outer cellular envelope of Chlamydia pneumoniae or one of its fragment, are preferred.

Among the said methods of preparing a polypeptide of the invention in recombinant form, the methods of preparation using a vector, and/or a cell transformed with the said vector and/or a transgenic animal comprising one of the said transformed cells, containing a nucleotide sequence encoding a Chlamydia pneumoniae secreted polypeptide or one of its representative fragments or encoding a transport polypeptide, a surface exposed polypeptide, a lipoprotein or one of its representative fragments, a polypeptide involved in lipopolysaccharide biosynthesis, a Type III or other secreted polypeptide, a polypeptide containing RGD attachment sites, a cell wall anchored surface polypeptide, a polypeptide not found in Chlamydia trachomatis, a ribosomal polypeptide or a polypeptide involved in secretion, transcription, translation, maturation of proteins, a polypeptide involved in the synthesis of the wall, a polypeptide involved in the virulence, a polypeptide involved in the intermediate metabolism, in particular in the metabolism of sugars and/or of cofactors, a polypeptide involved in the metabolism of nucleotides, of amino acids, of nucleic acids or of fatty acids of Chlamydia pneumoniae or one of their representative fragments, or a polypeptide specific to Chlamydia pneumoniae, are also preferred.

The recombinant polypeptides obtained as indicated above may be provided either in glycosylated or non-glycosylated form and may or may not have the natural tertiary structure.

A preferred variant consists in producing a recombinant polypeptide fused to a “carrier” protein (chimeric protein). The advantage of this system is that it allows a stabilization and a reduction in proteolysis of the recombinant product, an increase in solubility during renaturation in vitro and/or a simplification of purification when the fusion partner has affinity for a specific ligand.

More particularly, the invention relates to a method of preparing a polypeptide of the invention comprising the following steps:

a) culture of the transformed cells under conditions allowing the expression of a recombinant polypeptide having a nucleic acid sequence according to the invention;

b) where appropriate, recovery of the said recombinant polypeptide.

When the method of preparing a polypeptide of the invention uses a transgenic animal according to the invention, the recombinant polypeptide is then extracted from the said animal.

The subject of the invention is also a polypeptide capable of being obtained by a method of the invention as described above.

The invention also comprises a method of preparing a synthetic polypeptide, characterized in that it uses an amino acid sequence of polypeptides according to the invention.

The invention also relates to a synthetic polypeptide obtained by a method according to the invention.

Polypeptides according to the invention may also be prepared by conventional techniques in the field of peptide synthesis under conditions suitable to produce the polypeptides encoded by the polynucleotide of the invention.

This synthesis may be carried out in and recovered from a homogeneous solution or on a solid phase.

For example, the synthesis technique in a homogeneous solution described by Houbenweyl in 1974 may be used.

This method of synthesis consists in successively condensing, in pairs, the successive amino acids in the required order, or in condensing amino acids and fragments previously formed and already containing several amino acids in the appropriate order, or alternatively several fragments thus previously prepared, it being understood that care will have been taken to protect beforehand all the reactive functional groups carried by these amino acids or fragments, with the exception of the amine functional groups of one and the carboxyl functional groups of the other or vice versa, which should normally take part in the formation of the peptide bonds, in particular after activation of the carboxyl functional group, according to methods well known in peptide synthesis.

According to another preferred technique of the invention, the one described by Merrifield is used.

To manufacture a peptide chain according to the Merrifield method, a highly porous polymer resin is used, onto which the first C-terminal amino acid of the chain is attached. This amino acid is attached onto a resin via its carboxyl group and its amine functional group is protected. The amino acids which will constitute the peptide chain are thus attached, one after another, onto the amine group, each time deprotected beforehand, of the portion of the peptide chain already formed, and which is attached to the resin. When the entire peptide chain desired is formed, the protecting groups are removed from the various amino acids constituting the peptide chain and the peptide is detached from the resin with the aid of an acid.

The invention relates, in addition, to hybrid (fusion) polypeptides having at least one polypeptide or one of its representative fragments according to the invention, and a sequence of a polypeptide capable of eliciting an immune response in humans or animals.

Advantageously, the antigenic determinant is such that it is capable of eliciting a humoral and/or cellular response. An antigenic determinant may be identified by screening expression libraries of the Chlamydia pneumoniae genome with antibodies contained in the serum of patients infected with a bacterium belonging to the species Chlamydia pneumoniae. An antigenic determinant may comprise a polypeptide or one of its representative fragments according to the invention, in glycosylated form, used in order to obtain immunogenic compositions capable of inducing the synthesis of antibodies directed against multiple epitopes. The said polypeptides or their glycosylated fragments also form part of the invention.

These hybrid molecules may consist, in part, of a carrier molecule for polypeptides or for their representative fragments according to the invention, combined with a portion which may be immunogenic, in particular an epitope of the diphtheria toxin, the tetanus toxin, a hepatitis B virus surface antigen (patent FR 79 21811), the poliomyelitis virus VP1 antigen or any other viral or bacterial toxin or antigen.

The methods of synthesizing the hybrid molecules include the methods used in genetic engineering to construct hybrid nucleotide sequences encoding the desired polypeptide sequences. Reference may be advantageously made, for example, to the technique for producing genes encoding fusion proteins described by Minton in 1984.

The said hybrid nucleotide sequences encoding a hybrid polypeptide as well as the hybrid polypeptides according to the invention, characterized in that they are recombinant polypeptides obtained by the expression of the said hybrid nucleotide sequences, also form part of the invention.

The invention also comprises the vectors characterized in that they contain one of the said hybrid nucleotide sequences. The host cells transformed by the said vectors, the transgenic animals comprising one of the said transformed cells as well as the methods of preparing recombinant polypeptides using the said vectors, the said transformed cells and/or the said transgenic animals of course also form part of the invention.

The polypeptides according to the invention, the antibodies according to the invention described below and the nucleotide sequences according to the invention may advantageously be used in in vitro and/or in vivo methods for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae, in a biological sample (biological tissue or fluid) which is likely to contain them. These methods, depending on the specificity of the polypeptides, of the antibodies and of the nucleotide sequences according to the invention which will be used, may in particular detect and/or identify the bacterial variants belonging to the species Chlamydia pneumoniae as well as the associated microorganisms capable of being detected by the polypeptides, the antibodies and the nucleotide sequences according to the invention which will be chosen. It may, for example, be advantageous to choose a polypeptide, an antibody or a nucleotide sequence according to the invention, which is capable of detecting any bacterium of the Chlamydia family by choosing a polypeptide, an antibody and/or a nucleotide sequence according to the invention which is specific to the family or, on the contrary, it will be most particularly advantageous to target a variant of the species Chlamydia pneumoniae, which is responsible, for example, for the induction or the worsening of pathologies specific to the targeted variant, by choosing a polypeptide, an antibody and/or a nucleotide sequence according to the invention which is specific to the said variant.

The polypeptides according to the invention may advantageously be used in a method for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae or to an associated microorganism, in a biological sample (biological tissue or fluid) which is likely to contain them, characterized in that it comprises the following steps:

a) bringing this biological sample into contact with a polypeptide or one of its representative fragments according to the invention (under conditions allowing an immunological reaction between the said polypeptide and the antibodies which may be present in the biological sample);

b) detecting the antigen-antibody complexes which may be formed.

Preferably, the biological sample consists of a fluid, for example a human or animal serum, blood or biopsies.

Any conventional procedure may be used to carry out such a detection of the antigen-antibody complexes which may be formed.

By way of example, a preferred method uses immunoenzymatic procedures based on the ELISA technique, immunofluorescence procedures or radioimmunological procedures (RIA), and the like.

Accordingly, the invention also relates to the polypeptides according to the invention, labelled with the aid of a suitable label such as a label of the enzymatic, fluorescent or radioactive type.

Such methods comprise, for example, the following steps:

deposition of defined quantities of a polypeptide composition according to the invention into the wells of a microtitre plate,

introduction, into the said wells, of increasing dilutions of serum, or of a different biological sample as defined above, which has to be analysed,

incubation of the microplate,

introduction, into the wells of the microtitre plate, of labelled antibodies directed against human or animal immunoglobulins, these antibodies having been labelled with the aid of an enzyme selected from those which are capable of hydrolyzing a substrate, thereby modifying the absorption of the radiation of the latter, at least at a defined wavelength, for example at 550 nm,

detection, by comparison with a control, of the quantity of substrate hydrolyzed.

The invention also relates to a kit or set for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae or to an associated microorganism, characterized in that it comprises the following components:

a polypeptide according to the invention,

where appropriate, the reagents for constituting the medium appropriate for the immunological or specific reaction,

the reagents allowing the detection of the antigen-antibody complexes produced by the immunological reaction between the polypeptide(s) of the invention and the antibodies which may be present in the biological sample, it being possible for these reagents also to carry a label, or to be capable of being recognized in turn by a labelled reagent, more particularly in the case where the polypeptide according to the invention is not labelled,

where appropriate, a reference biological sample (negative control) free of antibodies recognized by a polypeptide according to the invention,

where appropriate, a reference biological sample (positive control) containing a predetermined quantity of antibodies recognized by a polypeptide according to the invention.

According to the invention, the polypeptides, peptides, fusion proteins or other derivatives, or analogs thereof encoded by a polynucleotide sequence in SEQ ID No. 1, may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen. Such antibodies may include, but are not limited to, polyclonal and monoclonal antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above. In a specific embodiment, the antibody to a polypeptide, peptide or other derivative, or analog thereof encoded by a polynucleotide sequence in SEQ ID No. 1 is a bispecific antibody (see generally, e.g. Fanger and Drakeman, 1995, Drug News and Perspectives 8: 133-137). Such a bispecific antibody is genetically engineered to recognize both (1) an epitope and (2) one of a variety of “trigger” molecules, e.g. Fc receptors on myeloid cells, and CD3 and CD2 on T cells, that have been identified as being able to cause a cytotoxic T-cell to destroy a particular target. Such bispecific antibodies can be prepared either by chemical conjugation, hybridoma, or recombinant molecular biology techniques known to the skilled artisan.

Various procedures known in the art may be used for the production of polyclonal antibodies to a polypeptide, peptide or other derivative, or analog thereof encoded by a polynucleotide sequence in SEQ ID No. 1. For the production of antibody, various host animals can be immunized by injection with a polypeptide, or peptide or other derivative, or analog thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants, depending on the host species, may be used to increase the immunological response, including but not limited to Stimulon™ QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, Mass.), MPL™ (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, Mont.), aluminum phosphate, IL-12 (Genetics Institute, Cambridge, Mass.), Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, BCG (bacille Calmette-Guerin), and corynebacterium parvum. Alternatively, polyclonal antibodies may be prepared by purifying, on an affinity column onto which a polypeptide according to the invention has been previously attached, the antibodies contained in the serum of patients infected with a bacterium belonging to the species Chlamydia pneumoniae.

For preparation of monoclonal antibodies directed toward a polypeptide, peptide or other derivative, or analog, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilizing technology described in PCT/US90/02545. In another embodiment of the invention, transgenic non-human animals can be used for the production of human antibodies utilizing technology described in WO 98/24893 and WO 96/33735. According to the invention, human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact, according to the invention, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, PROC. NATL. ACAD. SCI. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule specific for a polypeptide, peptide or other derivative, or analog together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention.

According to the invention, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce polypeptide or peptide-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for polypeptides, derivatives, or analogs.

Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)₂ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab′ fragments which can be generated by reducing the disulfide bridges of the F(ab′)₂ fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments.

In addition, techniques have been developed for the production of chimerized (See, e.g., Boss, M. et al., U.S. Pat. No. 4,816,397; and Cabilly, S. et al., U.S. Pat. No. 5,585,089 each of which is incorporated herein by reference in its entirety) humanized antibodies (See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated herein by reference in its entirety.) An immunoglobulin light or heavy chain variable region consists of a “framework” region interrupted by three hypervariable regions, referred to as complementarily determining regions (CDRs). The extent of the framework region and CDRs have been precisely defined (See, “Sequences of Proteins of Immunological Interest”, Kabat, E. et al., U.S. Department of Health and Human Services (1983). Briefly, humanized antibodies are antibody molecules from non-human species having one or more CDRs from the non-human species and a framework from a human immunoglobulin molecule.

The antibodies of the invention may also be labelled in the same manner as described above for the nucleic probes of the invention such as an enzymatic, fluorescent or radioactive type labelling.

The invention relates, in addition, to a method for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae or to an associated microorganism in a biological sample, characterized in that it comprises the following steps:

a) bringing the biological sample (biological tissue or fluid) into contact with a mono- or polyclonal antibody according to the invention (under conditions allowing an immunological reaction between the said antibodies and the polypeptides of the bacterium belonging to the species Chlamydia pneumoniae or to an associated microorganism which may be present in the biological sample, that is, under conditions suitable for the formation of immune complexes);

b) detecting the antigen-antibody complex which may be formed.

Also falling within the scope of the invention is a kit or set for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae or to an associated microorganism, characterized in that it comprises the following components:

a polyclonal or monoclonal antibody according to the invention, labeled where appropriate;

where appropriate, a reagent for constituting the medium appropriate for carrying out the immunological reaction;

a reagent allowing the detection of the antigen-antibody complexes produced by the immunological reaction, it being possible for this reagent also to carry a label, or to be capable of being recognized in turn by a labelled reagent, more particularly in the case where the said monoclonal or polyclonal antibody is not labelled;

where appropriate, reagents for carrying out the lysis of the cells in the sample tested.

The principle of the DNA chip which was explained above may also be used to produce protein “chips” on which the support has been coated with a polypeptide or an antibody according to the invention, or arrays thereof, in place of the DNA. These protein “chips” make it possible, for example, to analyze the biomolecular interactions (BIA) induced by the affinity capture of target analytes onto a support coated, for example, with proteins, by surface plasma resonance (SPR). Reference may be made, for example, to the techniques for coupling proteins onto a solid support which are described in EP 524 800 or to the methods describing the use of biosensor-type protein chips such as the BIAcore-type technique (Pharmacia) (Arlinghaus et al., 1997, Krone et al., 1997, Chatelier et al., 1995). These polypeptides or antibodies according to the invention, capable of specifically binding antibodies or polypeptides derived from the sample to be analysed, may thus be used in protein chips for the detection and/or the identification of proteins in samples. The said protein chips may in particular be used for infectious diagnosis and may preferably contain, per chip, several polypeptides and/or antibodies of the invention of different specificity, and/or polypeptides and/or antibodies capable of recognizing microorganisms different from Chlamydia pneumoniae.

Accordingly, the subject of the present invention is also the polypeptides and the antibodies according to the invention, characterized in that they are immobilized on a support, in particular of a protein chip.

The protein chips, characterized in that they contain at least one polypeptide or one antibody according to the invention immobilized on the support of the said chip, also form part of the invention.

The invention comprises, in addition, a protein chip according to the invention, characterized in that it contains, in addition, at least one polypeptide of a microorganism different from Chlamydia pneumoniae or at least one antibody directed against a compound of a microorganism different from Chlamydia pneumoniae, immobilized on the support of the said chip.

The invention also relates to a kit or set for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae or to an associated microorganism, or for the detection and/or the identification of a microorganism characterized in that it comprises a protein chip according to the invention.

The subject of the present invention is also a method for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae or to an associated microorganism in a biological sample, characterized in that it uses a nucleotide sequence according to the invention.

More particularly, the invention relates to a method for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae or to an associated microorganism in a biological sample, characterized in that it comprises the following steps:

a) where appropriate, isolation of the DNA from the biological sample to be analysed, or optionally production of a cDNA from the RNA in the biological sample;

b) specific amplification of the DNA of bacteria belonging to the species Chlamydia pneumoniae or to an associated microorganism with the aid of at least one primer according to the invention;

c) detection of the amplification products.

These may be detected, for example, by the molecular hybridization technique using a nucleic probe according to the invention. This probe will be advantageously labelled with a nonradioactive (cold probe) or radioactive element.

For the purposes of the present invention, “DNA in the biological sample” or “DNA contained in the biological sample” will be understood to mean either the DNA present in the biological sample considered, or optionally the cDNA obtained after the action of a reverse transcriptase-type enzyme on the RNA present in the said biological sample.

Another aim of the present invention consists in a method according to the invention, characterized in that it comprises the following steps:

a) bringing a nucleotide probe according to the invention into contact with a biological sample, the DNA contained in the biological sample having, where appropriate, been previously made accessible to hybridization, under conditions allowing the hybridization of the probe to complementary base pairs of the DNA of a bacterium belonging to the species Chlamydia pneumoniae or to an associated microorganism;

b) detecting the hybridization complex formed between the nucleotide probe and the DNA in the biological sample.

The present invention also relates to a method according to the invention, characterized in that it comprises the following steps:

a) bringing a nucleotide probe immobilized on a support according to the invention into contact with a biological sample, the DNA in the sample having, where appropriate, been previously made accessible to hybridization, under conditions allowing the hybridization of the probe to the DNA of a bacterium belonging to the species Chlamydia pneumoniae or to an associated microorganism;

b) bringing the hybrid formed between the nucleotide probe immobilized on a support and the DNA contained in the biological sample, where appropriate after removal of the DNA in the biological sample which has not hybridized with the probe, into contact with a labelled nucleotide probe according to the invention;

c) detecting the new hybrid formed in step b).

According to an advantageous embodiment of the method for the detection and/or the identification defined above, it is characterized in that, prior to step a), the DNA in the biological sample is primer-extended and/or amplified beforehand with the aid of at least one primer according to the invention.

The invention relates, in addition, to a kit or set for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae or to an associated microorganism, characterized in that it comprises the following components:

a) a nucleotide probe according to the invention;

b) where appropriate, the reagents necessary for carrying out a hybridization reaction;

c) where appropriate, at least one primer according to the invention as well as the reagents (e.g., polymerase and/or deoxynucleotide triphosphates) necessary for a DNA amplification reaction.

The invention also relates to a kit or set for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae or to an associated microorganism, characterized in that it comprises the following components:

a) a nucleotide probe, called capture probe, according to the invention;

b) an oligonucleotide probe, called detection probe, according to the invention;

c) where appropriate, at least one primer according to the invention as well as the reagents (e.g., polymerase and/or deoxynucleotide triphosphates) necessary for a DNA amplification reaction.

The invention also relates to a kit or set for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae or to an associated microorganism, characterized in that it comprises the following components:

a) at least one primer according to the invention;

b) where appropriate, the reagents necessary for carrying out a DNA amplification reaction;

c) where appropriate, a component which makes it possible to check the sequence of the amplified fragment, more particularly an oligonucleotide probe according to the invention.

The invention relates, in addition, to a kit or set for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae or to an associated microorganism, or for the detection and/or the identification of a microorganism characterized in that it comprises a DNA chip according to the invention.

The invention also relates to a method or to a kit or set according to the invention for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae, characterized in that the said primer and/or the said probe according to the invention are chosen from the nucleotide sequences specific to the species Chlamydia pneumoniae, in that the said polypeptides according to the invention are chosen from the polypeptides specific to the species Chlamydia pneumoniae and in that the said antibodies according to the invention are chosen from the antibodies directed against the polypeptides according to the invention chosen from the polypeptides specific to the species Chlamydia pneumoniae.

Preferably, the said method or the said kit or set above according to the invention, for the detection and/or the identification of bacteria belonging to the species Chlamydia pneumoniae is characterized in that the said primer and/or the said probe or the said polypeptides are chosen from the nucleotide sequences or polypeptides according to the invention which have been identified as being specific to the species Chlamydia pneumoniae and in that the said antibodies according to the invention are chosen from the antibodies directed against the polypeptides according to the invention chosen from the polypeptides identified as being specific to the species Chlamydia pneumoniae.

The invention relates, in addition, to a method or a kit or set according to the invention for the diagnosis of predispositions to, or of a condition caused by, cardiovascular diseases, preferably linked to the presence of atheroma, which are induced or worsened by a Chlamydia pneumoniae infection.

The invention also relates to a method or a kit or set according to the invention for the diagnosis of predispositions to, or of conditions caused by, respiratory diseases induced or worsened by a Chlamydia pneumoniae infection; preferably, the said respiratory disease is asthma.

According to another aspect, the subject of the invention is the use of polypeptides according to the invention, of cells transformed with a vector according to the invention and/or of transformed animals according to the invention, for the biosynthesis or the biodegradation of organic or inorganic compounds.

As has been mentioned above, the nucleotide sequences of the invention were identified by homology with sequences known to encode, for example, polypeptides or fragments of enzymatic polypeptides involved in the biosynthesis or the biodegradation of organic or inorganic molecules.

It is thus possible to use the said polypeptides of the invention in a similar manner for the biosynthesis or the biodegradation of organic or inorganic compounds of industrial or therapeutic interest (called compounds of interest).

Among these polypeptides, there may be mentioned in particular the enzymes involved in metabolism, such as the proteolytic enzymes, amino transferases, glucose metabolism, or the enzymes which may be used in the biosynthesis of sugars, amino acids, fatty acids, polypeptides, nucleotides, nucleic acids or any other organic or inorganic compound or in the biodegradation of organic or inorganic compounds.

Among these polypeptides, there may be mentioned, in addition, the mutated or modified enzymes corresponding to mutated or modified polypeptides according to the invention which may also be used for the biosynthesis or the biodegradation of organic or inorganic compounds at the industrial level, such as, for example, the production of compounds of interest, the reprocessing of manufacturing residues applied to the food industries, to the papermaking industry or to the chemical and pharmaceutical industries.

The methods of biosynthesis or biodegradation of organic or inorganic compounds, characterized in that they use a polypeptide or one of its representative fragments according to the invention, transformed cells according to the invention and/or a transformed animal according to the invention, also form part of the invention.

The invention relates, in addition, to the use of a nucleotide sequence according to the invention, of a polypeptide according to the invention, of an antibody according to the invention, of a cell according to the invention, and/or of a transformed animal according to the invention, for the selection of an organic or inorganic compound capable of modulating, regulating, inducing or inhibiting the expression of genes, and/or of modifying the cellular replication of eukaryotic or prokaryotic cells or capable of inducing, inhibiting or worsening the pathologies linked to an infection by Chlamydia pneumoniae or one of its associated microorganisms.

The invention also comprises screening assays that comprise methods of selecting compounds capable of binding to a polypeptide, fusion polypeptide or one of its representative fragments according to the invention, capable of binding to a nucleotide sequence according to the invention, or capable of recognizing an antibody according to the invention, and/or capable of modulating, regulating, inducing or inhibiting the expression of genes, and/or of modifying the growth or the cellular replication of eukaryotic or prokaryotic cells, or capable of inducing, inhibiting or worsening, in an animal or human organism, the pathologies linked to an infection by Chlamydia pneumoniae or one of its associated microorganisms, characterized in that it comprises the following steps:

a) bringing the said compound into contact with the said polypeptide, the said nucleotide sequence, with a transformed cell according to the invention and/or administering the said compound to a transformed animal according to the invention;

b) determining the capacity of the said compound to bind with the said polypeptide or the said nucleotide sequence, or to modulate, regulate, induce or inhibit the expression of genes, or to modulate growth or cellular replication, or to induce, inhibit or worsen in the said transformed animal, the pathologies linked to an infection by Chlamydia pneumoniae or one of its associated microorganisms.

The transformed cells and/or animals according to the invention may advantageously serve as a model and may be used in methods for studying, identifying and/or selecting compounds capable of being responsible for pathologies induced or worsened by Chlamydia pneumoniae, or capable of preventing and/or of treating these pathologies such as, for example, cardiovascular or respiratory diseases. In particular, the transformed host cells, in particular bacteria of the Chlamydia family whose transformation with a vector according to the invention may, for example, increase or inhibit its infectivity, or modulate the pathologies usually induced or worsened by the infection, may be used to infect animals in which the onset of pathologies will be monitored. These nontransformed animals, infected for example with transformed Chlamydia bacteria, may serve as a study model. In the same manner, the transformed animals according to the invention may, for example, exhibit predispositions to cardiovascular and/or respiratory diseases and thus be used in methods for selecting compounds capable of preventing and/or of treating the said diseases. The said methods using the said transformed cells and/or transformed animals form part of the invention.

The compounds capable of being selected may be organic compounds such as polypeptides or carbohydrates or any other organic or inorganic compounds already known, or new organic compounds produced using molecular modeling techniques and obtained by chemical or biochemical synthesis, these techniques being known to persons skilled in the art.

The said selected compounds may be used to modulate the growth and/or the cellular replication of Chlamydia pneumoniae or any other associated microorganism and thus to control infection by these microorganisms. The said compounds according to the invention may also be used to modulate the growth and/or the cellular replication of all eukaryotic or prokaryotic cells, in particular tumour cells and infectious microorganisms, for which the said compounds will prove active, the methods which make it possible to determine the said modulations being well known to persons skilled in the art.

Compound capable of modulating the growth of a microorganism is understood to designate any compound which makes it possible to act, to modify, to limit and/or to reduce the development, the growth, the rate of proliferation and/or the viability of the said microorganism.

This modulation may be achieved, for example, by an agent capable of binding to a protein and thus of inhibiting or of potentiating its biological activity, or capable of binding to a membrane protein of the outer surface of a microorganism and of blocking the penetration of the said microorganism into the host cell or of promoting the action of the immune system of the infected organism directed against the said microorganism. This modulation may also be achieved by an agent capable of binding to a nucleotide sequence of a DNA or RNA of a microorganism and of blocking, for example, the expression of a polypeptide whose biological or structural activity is necessary for the growth or for the reproduction of the said microorganism.

Associated microorganism is understood to designate in the present invention any microorganism whose gene expression may be modulated, regulated, induced or inhibited, or whose growth or cellular replication may also be modulated by a compound of the invention. Associated microorganism is also understood to designate in the present invention any microorganism containing nucleotide sequences or polypeptides according to the invention. These microorganisms may, in some cases, contain polypeptides or nucleotide sequences identical or homologous to those of the invention may also be detected and/or identified by the detection and/or identification methods or kit according to the invention and may also serve as a target for the compounds of the invention.

The invention relates to the compounds capable of being selected by a method of selection according to the invention.

The invention also relates to a pharmaceutical composition comprising a compound chosen from the following compounds:

a nucleotide sequence according to the invention;

a polypeptide according to the invention;

a vector according to the invention;

an antibody according to the invention; and

a compound capable of being selected by a method of selection according to the invention, optionally in combination with a pharmaceutically acceptable vehicle.

An effective quantity is understood to designate a sufficient quantity of the said compound or antibody, or of a polypeptide of the invention, which makes it possible to modulate the growth of Chlamydia pneumoniae or of an associated microorganism.

The invention also relates to a pharmaceutical composition comprising one or more polypeptides according to the invention and/or one or more fusion polypeptides according to the invention. Such compositions further comprise a pharmaceutically acceptable carrier or vehicle. Pharmaceutical compositions include compositions that comprise a polypeptide or fusion polypeptide that immunoreacts with seropositive serum of an individual infected with Chlamydia pneumoniae. In one embodiment, a pharmaceutical composition according to the invention can be utilized for the prevention or the treatment of an infection by a bacterium belonging to the species Chlamydia pneumoniae or by an associated microorganism.

The invention relates, in addition, to an immunogenic composition or a vaccine composition, characterized in that it comprises one or more polypeptides according to the invention and/or one or more hybrid (fusion) polypeptides according to the invention. Such compositions further comprise a pharmaceutically acceptable carrier or vehicle. Immunogenic compositions or fusion polypeptide include compositions that comprise a polypeptide that immunoreacts with seropositive serum of an individual infected with Chlamydia pneumoniae.

Immunogenic or vaccine compositions can also comprise DNA immunogenic or vaccine compositions comprising polynucleotide sequences of the invention operatively associated with a regulatory sequence that controls gene expression. Such compositions can include compositions that direct expression of a neutralizing epitope of Chlamydia pneumoniae.

The invention also comprises the use of a transformed cell according to the invention, for the preparation of a vaccine composition.

The invention also relates to a vaccine composition, characterized in that it contains a nucleotide sequence according to the invention, a vector according to the invention and/or a transformed cell according to the invention.

The invention also relates to the vaccine compositions according to the invention, for the prevention or the treatment of an infection by a bacterium belonging to the species Chlamydia pneumoniae or by an associated microorganism.

The invention also relates to the use of DNA encoding polypeptides of Chlamydia pneumoniae, in particular antigenic determinants, to be formulated as vaccine compositions. In accordance with this aspect of the invention, the DNA of interest is engineered into an expression vector under the control of regulatory elements, which will promote expression of the DNA, i.e., promoter or enhancer elements. In one preferred embodiment, the promoter element may be cell-specific and permit substantial transcription of the DNA only in predetermined cells. The DNA may be introduced directly into the host either as naked DNA (U.S. Pat. No. 5,679,647 incorporated herein by reference in their entirety) or formulated in compositions with other agents which may facilitate uptake of the DNA including viral vectors, i.e., adenovirus vectors, or agents which facilitate immunization, such as bupivicaine and other local anesthetics (U.S. Pat. No. 5,593,972 incorporated herein by reference in their entirety), saponins (U.S. Pat. No. 5,739,118 incorporated herein by reference in their entirety) and cationic polyamines (published international application WO 96/10038 incorporated herein by reference in their entirety).

The DNA sequence encoding the antigenic polypeptide and regulatory element may be inserted into a stable cell line or cloned microorganism, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described e.g., in Chappel, U.S. Pat. No. 4,215,051; Skoultchi, WO 91/06667 each of which is incorporated herein by reference in its entirety.

Such cell lines and microorganisms may be formulated for vaccine purposes. In yet another embodiment, the DNA sequence encoding the antigenic polypeptide and regulatory element may be delivered to a mammalian host and introduced into the host genome via homologous recombination (See, Chappel, U.S. Pat. No. 4,215,051; Skoultchi, WO 91/06667 each of which is incorporated herein by reference in its entirety.

Preferably, the immunogenic and/or vaccine compositions according to the invention intended for the prevention and/or the treatment of an infection by Chlamydia pneumoniae or by an associated microorganism will be chosen from the immunogenic and/or vaccine compositions comprising a polypeptide or one of its representative fragments corresponding to a protein, or one of its representative fragments, of the cellular envelope of Chlamydia pneumoniae. The vaccine compositions comprising nucleotide sequences will also preferably comprise nucleotide sequences encoding a polypeptide or one of its representative fragments corresponding to a protein, or one of its representative fragments, of the cellular envelope of Chlamydia pneumoniae.

Among these preferred immunogenic and/or vaccine compositions, the most preferred are those comprising a polypeptide or one of its representative fragments, or a nucleotide sequence or one of its representative fragments hose sequences are chosen from the nucleotide or amino acid sequences identified in this functional group and listed above.

The polypeptides of the invention or their representative fragments entering into the immunogenic compositions according to the invention may be selected by techniques known to persons skilled in the art, such as for example on the capacity of the said polypeptides to stimulate T cells, which results, for example, in their proliferation or the secretion of interleukins, and which leads to the production of antibodies directed against the said polypeptides.

In mice, in which a weight dose of the vaccine composition comparable to the dose used in humans is administered, the antibody reaction is tested by collecting serum followed by a study of the formation of a complex between the antibodies present in the serum and the antigen of the vaccine composition, according to the customary techniques.

According to the invention, the said vaccine compositions will be preferably in combination with a pharmaceutically acceptable vehicle and, where appropriate, with one or more appropriate immunity adjuvants.

Various types of vaccines are currently available for protecting humans against infectious diseases: attenuated live microorganisms (M. bovis—BCG for tuberculosis), inactivated microorganisms (influenza virus), acellular extracts (Bordetella pertussis for whooping cough), recombinant proteins (hepatitis B virus surface antigen), polysaccharides (pneumococci). Experiments are underway on vaccines prepared from synthetic peptides or from genetically modified microorganisms expressing heterologous antigens. Even more recently, recombinant plasmid DNAs carrying genes encoding protective antigens were proposed as an alternative vaccine strategy. This type of vaccination is carried out with a particular plasmid derived from an E. coli plasmid which does not replicate in vivo and which encodes only the vaccinal protein. Animals were immunized by simply injecting the naked plasmid DNA into the muscle. This technique leads to the expression of the vaccine protein in situ and to a cell-type (CTL) and a humoral type (antibody) immune response. This double induction of the immune response is one of the main advantages of the technique of vaccination with naked DNA.

The vaccine compositions of the present invention can be evaluated in in vitro and in vivo animal models prior to host, e.g., human, administration. For example, in vitro neutralization assays such as those described by Peterson et al. (1988) can be utilized. The assay described by Peterson et al. (1988) is suitable for testing vaccine compositions directed toward either Chlamydia pneumoniae or Chlamydia trachomatis.

Briefly, hyper-immune antisera is diluted in PBS containing 5% guinea pig serum, as a complement source. Chlamydiae (10⁴ IFU; inclusion-forming units) are added to the antisera dilutions. The antigen-antibody mixtures are incubated at 37° C. for 45 minutes and inoculated into duplicate confluent Hep-2 or HeLa cell monolayers contained in glass vials (e.g., 15 by 45 mm), which have been washed twice with PBS prior to inoculation. The monolayer cells are infected by centrifugation at 1000×g for 1 hour followed by stationary incubation at 37E for 1 hour. Infected monolayers are incubated for 48 or 72 hours, fixed and stained with a Chlamydiae specific antibody, such as anti-MOMP for C.trachomatis, etc. IFUs are counted in ten fields at a magnification of 200×. Neutralization titer is assigned based on the dilution that gives 50% inhibition as compared to control monolayers/IFU.

The efficacy of vaccine compositions can be determined in vivo by challenging animal models of Chlamydia pneumoniae infection, e.g., mice or rabbits, with the vaccine compositions. For example, in vivo vaccine composition challenge studies can be performed in the murine model of Chlamydia pneumonia infection described by Moazed et al. (1997). Briefly, male homozygous apoE deficient and/or C57 BL/6J mice are immunized with vaccine compositions. Post-vaccination, the mice are mildly sedated by subcutaneous injection of a mixture of ketamine and xylazine, and inoculated intranasally with a total volume of 0.03-0.05 ml of organisms suspended in SPG medium or with SPG alone. The inoculations of Chlamydia pneumoniae are approximately 3×10⁷ IFU/mouse. The mice are inoculated with Chlamydia pneumoniae at 8, 10, and 12 weeks of age. Tissues are then collected from the lung, spleen, heart, etc. at 1-20 weeks after the first inoculation. The presence of organisms is scored using PCR, histology and immunocytochemistry, or by quantitative culture/IFU after tissue homogenization.

Alternatively, in vivo vaccine composition challenge studies can be performed in the rabbit model of Chlamydia pneumoniae described by Laitinen et al. (1997). Briefly, New Zealand white rabbits (5 months old) are immunized with the vaccine compositions. Post-vaccination, the rabbits are sedated with Hypnorm, 0.3 ml/Kg of body weight, intramuscularly, and inoculated intranasally with a total of 0.5 ml of Chlamydia pneumoniae suspended in SPG medium or with SPG alone. The inoculations of Chlamydia pneumoniae are approximately 3×10⁷ IFU/rabbit. The rabbits are reinfected in the same manner and with the same dose 3 weeks after the primary inoculation. Tissues are then collected 2 weeks after the primary infection and 1, 2, and 4 weeks after the reinfection. The presence of Chlamydia pneumoniae is scored using PCR, histology and immunocytochemistry, or by quantitative culture/IFU after tissue homogenization.

The vaccine compositions comprising nucleotide sequences or vectors into which the said sequences are inserted are in particular described in International Application No. WO 90/11092 and also in International Application No. WO 95/11307.

The nucleotide sequence constituting the vaccine composition according to the invention may be injected into the host after having been coupled to compounds which promote the penetration of this polynucleotide inside the cell or its transport up to the cell nucleus. The resulting conjugates may be encapsulated into polymeric microparticles, as described in International Application No. WO 94/27238 (Medisorb Technologies International).

According to another embodiment of the vaccine composition according to the invention, the nucleotide sequence, preferably a DNA, is complexed with the DEAE-dextran (Pagano et al., 1967) or with nuclear proteins (Kaneda et al., 1989), with lipids (Felgner et al., 1987) or encapsulated into liposomes (Fraley et al., 1980) or alternatively introduced in the form of a gel facilitating its transfection into the cells (Midoux et al., 1993, Pastore et al., 1994). The polynucleotide or the vector according to the invention may also be in suspension in a buffer solution or may be combined with liposomes.

Advantageously, such a vaccine will be prepared in accordance with the technique described by Tacson et al. or Huygen et al. in 1996 or alternatively in accordance with the technique described by Davis et al. in International Application No. WO 95/11307.

Such a vaccine may also be prepared in the form of a composition containing a vector according to the invention, placed under the control of regulatory elements allowing its expression in humans or animals. It is possible, for example, to use, as vector for the in vivo expression of the polypeptide antigen of interest, the plasmid pcDNA3 or the plasmid pcDNA1/neo, both marketed by Invitrogen® & D Systems, Abingdon, United Kingdom). It is also possible to use the plasmid V1Jns.tPA, described by Shiver et al. in 1995. Such a vaccine will advantageously comprise, in addition to the recombinant vector, a saline solution, for example a sodium chloride solution.

The immunogenic compositions of the invention can also be utilized as part of methods for immunization, wherein such methods comprise administering to a host, e.g., a human host, an immunizing amount of the immunogenic compositions of the invention. In a preferred embodiment, the method of immunizing is a method of immunizing against Chlamydia pneumoniae.

A pharmaceutically acceptable vehicle is understood to designate a compound or a combination of compounds entering into a pharmaceutical or vaccine composition which does not cause side effects and which makes it possible, for example, to facilitate the administration of the active compound, to increase its life and/or its efficacy in the body, to increase its solubility in solution or alternatively to enhance its preservation. These pharmaceutically acceptable vehicles are well known and will be adapted by persons skilled in the art according to the nature and the mode of administration of the active compound chosen.

As regards the vaccine formulations, these may comprise appropriate immunity adjuvants which are known to persons skilled in the art, such as, for example, aluminum hydroxide, a representative of the family of muramyl peptides such as one of the peptide derivatives of N-acetyl-muramyl, a bacterial lysate, or alternatively incomplete Freund's adjuvant, Stimulon™ QS-21 (Aquila Biopharmaceuticals, Inc., Framingham, Mass.), MPL™ (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem Research, Inc., Hamilton, Mont.), aluminum phosphate, IL-12 (Genetics Institute, Cambridge, Mass.).

Preferably, these compounds will be administered by the systemic route, in particular by the intravenous route, by the intranasal, intramuscular, intradermal or subcutaneous route, or by the oral route. More preferably, the vaccine composition comprising polypeptides according to the invention will be administered several times, spread out over time, by the intradermal or subcutaneous route.

Their optimum modes of administration, dosages and galenic forms may be determined according to criteria which are generally taken into account in establishing a treatment adapted to a patient, such as for example the patient's age or body weight, the seriousness of his general condition, tolerance of the treatment and the side effects observed.

The invention comprises the use of a composition according to the invention for the treatment or the prevention of cardiovascular diseases, preferably linked to the presence of atheroma, which are induced or worsened by Chlamydia pneumoniae.

Finally, the invention comprises the use of a composition according to the invention for the treatment or the prevention of respiratory diseases which are induced or worsened by the presence of Chlamydia pneumoniae, preferably asthma.

Other characteristics and advantages of the invention appear in the following examples and figures:

LEGEND TO THE FIGURES

FIG. 1: Line for the production of Chlamydia pneumoniae sequences

FIG. 2: Analysis of the sequences and assembling

FIG. 3: Finishing techniques

FIG. 3a): Assembly map

FIG. 3b): Determination and use of the orphan ends of the contigs

EXAMPLES

Experimental Procedures

Cells

The Chlamydia pneumoniae strain (CM1) used by the inventors is obtained from ATCC (American Culture Type Collection) where it has the reference number ATCC 1360-VR.

It is cultured on HeLa 229 cells, obtained from the American Type Culture Collection, under the reference ATCC CCL-2.1.

Culture of the Cells

The HeLa ATCC CCL-2.1 cells are cultured in 75-ml cell culture flasks (Corning). The culture medium is Dulbecco's modified cell culture medium (Gibco BRL No. 04101965) supplemented with MEM amino acids (Gibco BRL—No. 04301140) L (5 ml per 500 ml of medium) and 5% foetal calf serum (Gibco BRL No. 10270 batch 40G8260K) without antibiotics or antifungals.

The cell culture stock is maintained in the following manner. The cell cultures are examined under an inverted microscope. 24 hours after confluence, each cellular lawn is washed with PBS (Gibco BRL No. 04114190), rinsed and then placed for 5 min in an oven in the presence of 3 ml of trypsine (Gibco BRL No. 25200056). The cellular lawn is then detached and then resuspended in 120 ml of culture medium, the whole is stirred in order to make the cellular suspension homogeneous. 30 ml of this suspension are then distributed per cell culture flask. The flasks are kept in a CO₂ oven (5%) for 48 hours at a temperature of 37° C. The cell stock is maintained so as to have available daily 16 flasks of subconfluent cells. It is these subconfluent cells which will be used so as to be infected with Chlamydia. 25-ml cell culture flasks are also used, these flasks are prepared in a similar manner but the volumes used for maintaining the cells are the following: 1 ml of trypsine, 28 ml of culture medium to resuspend the cells, 7 ml of culture medium are used per 25-ml flask.

Infection of the Cells with Chlamydia

Initially, the Chlamydiae are obtained frozen from ATCC (−70° C.), in suspension in a volume of 1 ml. This preparation is slowly thawed, 500 ml are collected and brought into contact with subconfluent cells, which are obtained as indicated above, in a 25-ml cell culture flask, containing 1 ml of medium, so as to cover the cells. The flask is then centrifuged at 2000 rpm in a “swing” rotor for microtitre plates, the centrifuge being maintained at a temperature of 35° C. After centrifugation, the two flasks are placed in an oven at 35° C. for three hours. 6 ml of culture medium containing cycloheximide (1 mg/ml) are then added and the flask is stored at 35° C. After 72 hours, the level of infection is evaluated by direct immunofluorescence and by the cytopathogenic effect caused to the cells.

Direct Immunofluorescence

Starting with infected cells, which were obtained as indicated above, a cellular smear is deposited with a Pasteur pipette on a microscope slide. The cellular smear is fixed with acetone for 10 minutes; after draining the acetone, the smear is covered with 30 ml of murine monoclonal antibodies directed against MOMP (major outer membrane protein) of Chlamydia (Syva, Biomérieux) labelled with fluorescein isothiocyanate. The whole is then incubated in a humid chamber at a temperature of 37° C. The slides are then rinsed with water, slightly dried, and then after depositing a drop of mounting medium, a coverslip is mounted before reading. The reading is carried out with the aid of a fluorescence microscope equipped with the required filters (excitation at 490 nm, emission at 520 nm).

Harvesting of the Chlamydia pneumoniae

After checking the infection by direct immunofluorescence, carried out as indicated above, the culture flasks are opened under a sterile cabinet, sterile glass beads with a diameter of the order of a millimeter are placed in the flask. The flask is closed and then vigorously stirred while being maintained horizontally, the cellular lawn at the bottom, so that the glass beads can have a mechanical action on the cellular lawn. Most of the cells are thus detached or broken; the effect of the stirring is observed under an optical microscope so as to ensure proper release of Chlamydiae.

Large-scale Infection of the Cell Cultures

The product of the Chlamydiae harvest (culture medium and cellular debris) is collected with a pipette, and distributed into three cell culture flasks containing subconfluent HeLa ATCC CCL-2.1 cells, obtained as indicated above. The cells thus inoculated are placed under gentle stirring (swing) in an oven at 35° C. After one hour, the flasks are kept horizontally in an oven so that the culture medium covers the cells for 3 hours. 30 ml of culture medium containing actydione (1 mg/ml) are then added to each of the flasks. The culture flasks are then stored at 35° C. for 72 hours. The cells thus infected are examined under an optical microscope after 24 hours, the cytopathogenic effect is evaluated by the appearance of cytoplasmic inclusions which are visible under an inverted optical microscope. After 72 hours, the vacuoles containing the Chlamydiae occupy the cytoplasm of the cell and push the cell nucleus sideways. At this stage, numerous cells are spontaneously destroyed and have left free elementary bodies in the culture medium. The Chlamydiae are harvested as described above and are either frozen at −80° C. or used for another propagation.

Purification of the Chlamydiae

The product of the Chlamydia harvests is stored at −80° C. and thawed on a water bath at room temperature. After thawing, each tube is vigorously stirred for one minute and immersed for one minute in an ultrasound tank (BRANSON 1200); the tubes are then stirred by inverting before being centrifuged for 5 min at 2000 rpm. The supernatant is carefully removed and kept at cold temperature (ice). The supernatant is vigorously stirred and then filtered on nylon filters having pores of 5 microns in diameter on a support (Nalgene) allowing a delicate vacuum to be established under the nylon filter. For each filtration, three nylon filters are superposed; these filters are replaced after every 40 ml of filtrate. Two hundred milliliters of filtration product are kept at cold temperature, and then after stirring by inverting, are centrifuged at 10,000 rpm for 90 min, the supernatant is removed and the pellet is taken up in 10 ml of 10 mM Tris, vigorously vortexed and then centrifuged at 10,000 rpm for 90 min. The supernatant is removed and the pellet is taken up in a buffer (20 mM Tris pH 8.0, 50 mM KCl, 5 mM MgCl₂) to which 800 units of DNAse I (Boehringer) are added. The whole is kept at 37° C. for one hour. One ml of 0.5 M EDTA is then added, the whole is vortexed and frozen at −20° C.

Preparation of the DNA

The Chlamydiae purified above are thawed and subjected to a proteinase K (Boehringer) digestion in a final volume of 10 ml. The digestion conditions are the following: 0.1 mg/ml proteinase K, 0.1×SDS at 55° C., stirring every 10 min. The product of digestion is then subjected to a double extraction with phenol-chloroform, two volumes of ethanol are added and the DNA is directly recovered with a Pasteur pipette having one end in the form of a hook. The DNA is dried on the edge of the tube and then resuspended in 500 ml of 2 mM Tris pH 7.5. The DNA is stored at 4° C. for at least 24 hours before being used for the cloning.

Cloning of the DNA

After precipitation, the DNA is quantified by measuring the optical density at 260 nm. Thirty mg of Chlamydia DNA are distributed into 10 tubes of 1.5 ml and diluted in 300 ml of water. Each of the tubes is subjected to 10 applications of ultrasound lasting for 0.5 sec in a sonicator (unisonix XL2020). The contents of the 10 tubes are then grouped and concentrated by successive extractions with butanol (Sigma B1888) in the following manner: two volumes of butanol are added to the dilute DNA mixture. After stirring, the whole is centrifuged for five minutes at 2500 rpm and the butanol is removed. This operation is repeated until the volume of the aqueous phase is less than 1 ml. The DNA is then precipitated in the presence of ethanol and of 0.5 M sodium acetate pH 5.4, and then centrifuged for thirty minutes at 15,000 rpm at cold temperature (4° C.). The pellet is washed with 75% ethanol, centrifuged for five minutes at 15,000 rpm and dried at room temperature. A tenth of the preparation is analysed on a 0.8% agarose gel. Typically, the size of the DNA fragments thus prepared is between 200 and 8000 base pairs.

To allow the cloning of the DNA obtained, the ends are repaired. The DNA is distributed in an amount of 10 mg/tube, in the following reaction medium: 100 ml final volume, 1×buffer (Biolabs 201L), 0.5 ml BSA 0.05 mg/ml, 0.1 mM DATP, 0.1 mM each of dGTP, dCTP or dTTP, 60,000 IU T4 DNA polymerase. The reaction is incubated for thirty minutes at 16° C. The contents of each of the tubes are then grouped before carrying out an extraction with phenol-chloroform and then precipitating the aqueous phase as described above. After this step, the DNA thus prepared is phosphorylated. For that, the DNA is distributed into tubes in an amount of 10 mg per tube, and then in a final volume of 50 ml, the reaction is prepared in the following manner: 1 mM ATP, 1×kinase buffer, 10 IU T4 polynucleotide kinase (Biolabs 201L). The preparation is incubated for thirty minutes at 37° C. The contents of the tubes are combined and a phenol-chloroform extraction and then a precipitation are carried out in order to precipitate the DNA. The latter is then suspended in 1 ml of water and then the DNA fragments are separated according to their size on a 0.8% agarose gel (1×TAE). The DNA is subjected to an electric field of 5 V/cm and then visualized on a UV table. The fragments whose size varies between 1200 and 2000 base pairs are selected by cutting out the gel. The gel fragment thus isolated is placed in a tube and then the DNA is purified with the Qiaex kit (20021 Qiagen), according to the procedure provided by the manufacturer.

Preparation of the Vector

14 mg of the cloning vector pGEM-5Zf (Proméga P2241) are diluted in a final volume of 150 ml and are subjected to digestion with the restriction enzyme EcoRV 300 IU (Biolabs 195S) according to the protocol and with the reagents provided by the manufacturer. The whole is placed at 37° C. for 150 min and then distributed in the wells of a 0.8% agarose gel subjected to an electric field of 5 V/cm. The linearized vector is visualized on a UV table, isolated by cutting out the gel and then purified by the Qiaex kit (Qiagen 20021) according to the manufacturer's recommendations. The purification products are grouped in a tube, the volume is measured and then half the volume of phenol is added and the whole is vigorously stirred for 1 min. Half the volume of chloroform-isoamyl alcohol 24:1 is added and vigorously stirred for 1 min. The whole is centrifuged at 15,000 rpm for 5 min at 4° C., the aqueous phase is recovered and transferred into a tube. The DNA is precipitated in the presence of 0.3 M sodium acetate, pH 5.4 and 3 volumes of ethanol and placed at −20° C. for 1 hour. The DNA is then centrifuged at 15,000 rpm for 30 min at 4° C., the supernatant is removed while preserving the pellet, washed twice with 70% ethanol. After drying at room temperature, the DNA is suspended in 25 ml of water.

Phosphorylation of the Vector

25 ml of the vector prepared in the preceding step are diluted in a final volume of 500 ml of the following reaction mixture:

After repair, the DNA is subjected to a phenol-chloroform extraction and a precipitation, the pellet is then taken up in 10 ml of water, the DNA is quantified by measuring the optical density at 260 nm. The quantified DNA is ligated into the vector PGEm-5Zf(+) prepared by the restriction enzyme EcoRV and dephosphorylated (see preparation of the vector). The ligation is carried out under three conditions which vary in the ratio between the number of vector molecules and the number of insert molecules. Typically, an equimolar ratio, a ratio of 1:3 and a ratio of 3:1 are used for the ligations which are, moreover, carried out under the following conditions: vector PGEm-5Zf(+) 25 ng, cut DNA, ligation buffer in a final volume of 20 ml with T4 DNA ligase (Amersham E70042X); the whole is then placed in a refrigerator overnight and then a phenol-chloroform extraction and a precipitation are carried out in a conventional manner. The pellet is taken up in 5 ml of water.

Transformation of the Bacteria

Plating of the Bacteria

Petri dishes containing LB Agar medium containing ampicillin (50 mg/ml), Xgal (280 mg/ml) [5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (Sigma B-4252)], IPTG (140 mg/ml) [isopropyl-beta-D-thiogalactoside (Sigma I-6758)] are used, 50 and 100 ml of bacteria are plated for each of the ligations. The Petri dishes are placed upside down at 37° C. for 15 to 16 hours in an oven. The number of “recombinant” positive clones is evaluated by counting the white colonies and the blue colonies which are thought to contain the vector alone.

Evaluation of the “recombinant” positive clones

Ninety-four white colonies and two blue colonies are collected with the aid of sterile cones and are deposited at the bottom of the wells of plates designed for carrying out the amplification techniques. 30 ml of the following reaction mixture are added to each well: 1.7 mM MgCl₂, 0.2 mM each of dTP, dCTP, dGTP and dTTP, two synthetic oligonucleotides corresponding to sequences flanking the cloning site on either side and orienting the synthesis of the DNA in a convergent manner (0.5 mM RP and PU primers, 1 U TAQ polymerase (GibcoBRL 18038-026)).

The colonies thus prepared are subjected to a temperature of 94° C. for 5 min and then to 30 thermal cycles composed of the following steps: 94° C. for 40 s, 50° C. for 30 s, 72° C. for 180 s. The reaction is then kept for 7 min at 72° C. and then kept at 4° C.

The amplification products are deposited on an agarose gel (0.8%), stained with ethidium bromide, subjected to electrophoresis, and then analysed on an ultraviolet table. The presence of an amplification fragment having a size greater than 500 base pairs indicates the presence of an insert. The bacterial clones are then prepared so as to study the sequence of their insert.

Sequencing

To sequence the inserts of the clones obtained as above, these were amplified by PCR on bacteria cultures carried out overnight using the primers for the vectors flanking the inserts. The sequence of the ends of these inserts (on average 500 bases on each side) was determined by automated fluorescent sequencing on an ABI 377 sequencer, equipped with the ABI Prism DNA Sequencing Analysis software (version 2.1.2).

Analysis of the Sequences

The sequences obtained by sequencing in a high-yield line (FIG. 1) are stored in a database; this part of the production is independent of any treatment of the sequences. The sequences are extracted from the database, avoiding all the regions of inadequate quality, that is to say the regions for which uncertainties are observed on the sequence at more than 95%. After extraction, the sequences are introduced into a processing line, the diagram of which is described in FIG. 2. In a first path of this processing line, the sequences are assembled by the Gap4 software from R. Staden (Bonfield et al., 1995) (OS UNIX/SUN Solaris); the results obtained by this software are kept in the form of two files which will be used for a subsequent processing. The first of these files provides information on the sequence of each of the contigs obtained. The second file represents all the clones participating in the composition of all the contigs as well as their positions on the respective contigs.

The second processing path uses a sequence assembler (TIGR-Asmg assembler UNIX/SUN Solaris); the results of this second processing path are kept in the form of a file in the TIGR-Asmg format which provides information on the relationship existing between the sequences selected for the assembly. This assembler is sometimes incapable of linking contigs whose ends overlap over several hundreds of base pairs.

The results obtained from these two assemblers are compared with the aid of the BLAST program, each of the contigs derived from one assembly path being compared with the contigs derived from the other path.

For the two processing paths, the strict assembly parameters are fixed (95% homology, 30 superposition nucleotides). These parameters avoid 3 to 5% of the clones derived from eukaryotic cells being confused with sequences obtained from the clones derived from Chlamydia pneumoniae. The eukaryotic sequences are however preserved during the course of this project; the strategy introduced, which is described below, will be designed, inter alia, not to be impeded by these sequences derived from contaminating clones.

The results of these two assemblers are processed in a software developed for this project. This software operates on a Windows NT platform and receives, as data, the results derived from the STADEN software and/or the results derived from the TIGR-Asmg assembler, the software, results, after processing of the data, in the determination of an assembly map which gives the proximity relationship and the orientation of the contigs in relation to one another (FIG. 3a). Using this assembly map, the software determines all the primers necessary for finishing the project. This treatment, which will be detailed below, has the advantage of distinguishing the isolated sequences derived from the contaminations, by the DNA eukaryotic cells, of the small-sized sequences clearly integrated into the project by the relationships which they establish with contigs. In order to allow, without any risk of error, the arrangement and the orientation of the contigs in relation to one another, a statistical evaluation of the accuracy of the names (naming) “naming” of sequence is made from the results of “contigation”. This evaluation makes it possible to give each of the clone plates, as well as each of the subsets of plates, a weight which is inversely proportional to probable error rate existing in the “naming” of the sequences obtained from this plate or from a subset of this plate. In spite of a low error rate, errors may occur throughout the steps of production of the clones and of the sequences. These steps are numerous, repetitive and although most of them are automated, others, like the deposition in the sequencers, are manual; it is then possible for the operator to make mistakes such as the inversion of two sequences. This type of error has a repercussion on the subsequent processing of the data, by resulting in relationships (between the contigs) which do not exist in reality, then in attempts at directed sequencing between the contigs which will end in failure. It is because of this that the evaluation of the naming errors is of particular importance since it allows the establishment of a probabilistic assembly map from which it becomes possible to determine all the clones which will serve as template to obtain sequences separating two adjacent contigs. Table 2 of parent U.S. application serial No. 60/107078 filed Nov. 4, 1998 and French application 97-14673 filed Nov. 21, 1997, each of which is incorporated by reference herein in its entirety, gives the clones and the sequences of the primers initially used during the initial operations.

To avoid the step which consists in ordering and then preparing the clones by conventional microbiological means, outer and inner primers oriented towards the regions not yet sequenced are defined by the software. The primers thus determined make it possible to prepare, by PCR, a template covering the nonsequenced region. It is the so-called outer primers (the ones most distant from the region to be sequenced) which are used to prepare this template. The template is then purified and a sequence is obtained on each of the two strands during 2 sequencing reactions which each use one of the 2 inner primers. In order to facilitate the use of this approach, the two outer primers and the two inner primers are prepared and then stored on the same position of 4 different 96-well plates. The two plates containing the outer primers are used to perform the PCRs which will serve to prepare the templates. These templates will be purified on purification columns preserving the topography of the plates. Each of the sequences will be obtained using primers situated on one and then on the other of the plates containing the inner primers. This distribution allows a very extensive automation of the process and results in a method which is simple to use for finishing the regions not yet sequenced. Table 3 of parent U.S. application serial No. 60/107078 filed Nov. 4, 1998 and French application 97-14673 filed Nov. 21, 1997, each of which is incorporated by reference herein in its entirety, gives the names and the sequences of the primers used for finishing Chlamydia pneumoniae.

Finally, a number of contigs exist in a configuration where one of their ends is not linked to any other contig end (FIG. 3b) by a connecting clone relationship (a connecting clone is defined as a clone having one sequence end on a contig and the other end of its sequence on another contig; furthermore, this clone must be derived from a plate or a subset of plates with adequate naming quality). For the Chlamydia pneumoniae project, this particular case occurred 24 times. Two adjacent PCR primers orienting the synthesis of the DNA towards the end of the consensus sequence are defined for each of the orphan ends of the consensus sequence. The primer which is closest to the end of the sequence is called the inner primer whereas the primer which is more distant from the end of the sequence is called the outer primer. The outer primers are used to explore the mutual relationship between the orphan ends of the different contigs. The presence of a single PCR product and the possibility of amplifying this product unambiguously using the inner primers evokes the probable relationship between the contigs on which the primers which allowed the amplification are situated. This relationship will be confirmed by sequencing and will allow the connection between the orphan ends of the consensus sequences. This strategy has made it possible to obtain a complete map of the Chlamydia pneumoniae chromosome and then to finish the project.

Quality control

All the bases not determined with certainty in the chromosomal sequence were noted and the density of uncertainties was measured on the entire chromosome. The regions with a high density of uncertainties were noted and the PCR primers spanning these regions were drawn and are represented in Table 4 of parent U.S. application serial No. 60/107078 filed Nov. 4, 1998 and French application 97-14673 filed Nov. 21, 1997 each of which is incorporated by reference herein in its entirety.

The sequence of each of the PCR products was obtained with two operational primers different from the amplification primers. The sequences were obtained in both directions for all the PCRs (100% success).

Data banks

Local reorganizations of major public banks were used. The protein bank used consists of the nonredundant fusion of the Genpept bank (automated translation of GenBank, NCBI; Benson et al., 1996).

The entire BLAST software (public domain, Altschul et al., 1990) for searching for homologies between a sequence and protein or nucleic data banks was used. The significance levels used depend on the length and the complexity of the region tested as well as the size of the reference bank. They were adjusted and adapted to each analysis.

The results of the search for homologies between a sequence according to the invention and protein or nucleic data banks are presented and summarized in Table 1 below.

Table 1: List of Coding Chromosome Regions and Homologies Between These Regions and the Sequence Banks

Legend to Table 1: Open reading frames are identified with the GenMark software version 2.3A (GenePro), the template used is Chlamydia pneumoniae of order 4 on a length of 196 nucleotides with a window of 12 nucleotides and a minimum signal of 0.5. The reading frames ORF2 to ORF 1137 are numbered in order of appearance on the chromosome, starting with ORF2 (ORF column). The positions of the beginning and of the end are then given in column 2 (position). When the position of the beginning is greater than the position of the end, this means that the region is encoded by the strand complementary to the sequence which was given in the sequence SEQ ID No. 1.

All the putative products were subjected to a search for homology on GENPEPT (release 102 for SEQ ID No. 2 to SEQ ID No. 1137, and release 108 for SEQ ID No. 1138 to SEQ ID No. 1291 and SEQ ID No. 6844 to SEQ ID No. 6849) with the BLASTP software (Altschul et al. 1990). With, as parameters, the default parameters with the exception of the expected value E set at 10⁻⁵ (for SEQ ID No. 2 to SEQ ID No. 1137) and P value set at e⁻¹⁰ (for SEQ ID No. 1138 to SEQ ID No. 1291 and SEQ ID No. 6844 to SEQ ID No. 6849). Subsequently, only the identities greater than 30% (I% column) were taken into account. The description of the most homologous sequence is given in the Homology column; the identifier for the latter sequence is given in the ID column and the animal species to which this sequence belongs is given in the Species column. The Homology score is evaluated by the sum of the blast scores for each region of homology and reported in the Score column.

Materials and Methods for transmembrane domains:

The DAS software was used as recommended by the authors (Cserzo et al., 1997).

This method uses, to predict the transmembrane domains, templates derived from a sampling of selected proteins. All the regions for which a “Cutoff” greater than 1.5 was found by the program were taken into account.

Additional ORF Finder Programs

For this analysis, two additional ORF finder programs were used to predict potential open reading frames of a minimum length of 74 amino acids; Glimmer (Salzberg, S. L., Delcher, A., Kasif, S., and W. White. 1998. Microbial gene identification using interpolated Markov models. Nucleic Acids Res. 26:544-548.), and an in-house written program. The in-house program used a very simple search algorithm. The analysis required the that the genomic DNA sequence text be in the 5′ to 3′ direction, the genome is circular, and that TAA, TAG, and TGA are stop codons. The search parameters were as follows:

(1) A search for an ORF that started with a GTG codon was performed. If no GTG codons were found, then a search for an ATG codon was performed. However, if a GTG codon was found, then a search downstream for a ATG codon was performed. All start and stop nucleotide positions were recorded.

(2) A search for an ORF that started with a TTG codon was performed. If no TTG codons were found, then a search for a ATG codon was performed. However, if a TTG codon was found, then a search downstream for a ATG codon was performed. All start and stop nucleotide positions were recorded.

(3) The analysis described in steps 1 and 2 were repeated for the opposite strand of DNA sequence.

(4) A search for ORFs that determined all ORF lengths using start and stop positions in the same reading frames was performed.

(5) All ORFs whose DNA length was less than 225 nucleotides were eliminated from the search.

Surface Exposed Protein Search Criteria

Potential cell surface vaccine targets are outer membrane proteins such as porins, lipoproteins, adhesions and other non-integral proteins. In Chlamydia psittaci, the major immunogens is a group of putative outer membrane proteins (POMPs) and no homologs have been found in Chlamydia pneumoniae and Chlamydia trachomatis by traditional analysis (Longbottom, D., Russell, M., Dunbar, S. M., Jones, G. E., and A. J. Herring. 1998. Molecular Cloning and Characterization of the Genes Coding for the Highly Immunogenic Cluster of 90-Kilodalton Envelope Proteins from Chlamydia psittaci Subtype That Causes Abortion in Sheep. Infect Immun 66:1317-1324.) Several putative outer membrane proteins have been identified in Chlamydia pneumoniae, all of which may represent vaccine candidates. The major outer membrane protein (MOMP) gene (omp1) has been found in various isolates of Chlamydia pneumoniae (Jantos, Calif., Heck, S., Roggendorf, R., Sen-Gupta, M., and Hegemann, J H. 1997. Antigenic and molecular analyses of different chlamydia pneumoniae strains. J. Clin Microbiology 35(3):620-623.) Various criteria, as listed below, were used to identify putative surface exposed ORFs from the genomic DNA sequence of Chlamydia pneumoniae (French application 97-14673 filed Nov. 21, 1997). Any ORF which met any one or more of the individual criteria were listed in this category.

Protein homology searches were done using the Blastp 2.0 tool (Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.)

An ORF product was labeled surface exposed if there was homology to a known, or hypothetical, or putative surface exposed protein with a P score better than e⁻¹⁰.

Most, if not all, proteins that are localized to the membrane of bacteria, via a secretory pathway, contain a signal peptide. A software program, SignalP, analyzes the amino acid sequence of an ORF for such a signal peptide (Nielsen, H., Engelbrecht. J., Brunak, S., and G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineering 10:1-6.) The first 60 N-terminal amino acids of each ORF were analyzed by SignalP using the Gram-Negative software database. The output generates four separate values, maximum C, maximum Y, maximum S, and mean S. The S-score, or signal region, is the probability of the position belonging to the signal peptide. The C-score, or cleavage site, is the probability of the position being the first in the mature protein. The Y-score is the geometric average of the C-score and a smoothed derivative of the S-score. A conclusion of either a Yes or No is given next to each score. If all four conclusions are Yes and the C-terminal amino acid is either a phenylalanine (F) or a tyrosine (Y), the ORF product was labelled outer membrane (Struyve, M., Moons, M., and J. Tommassen. 1991. Carboxy-terminal Phenylalanine is Essential for the Correct Assembly of a Bacterial Outer Membrane Protein. J. Mol. Biol. 218:141-148.)

The program called Psort, determines the localization of a protein based on its signal sequence, recognition of transmembrane segments, and analysis of its amino acid composition (Nakai, K., and M. Kanehisa. 1991. Expert system for predicting protein localization sites in gram-negative bacteria. Proteins 11:95-110.) An ORF product is considered to be an outer membrane protein if the output data predicts the protein as outer membrane with a certainty value of 0.5 or better and whose value is at least twice as large as the next predicted localized certainty value.

Finally, ORF products that were not predicted to be outer membrane or surface exposed, based on the above criteria, were further analyzed. The blastp output data for these ORFs were searched using various general and specific keywords, suggestive of known cell surface exposed proteins. An ORF was labeled surface exposed if the keywords matched had a Blastp hit, had a P score better than e⁻¹⁰, and that there was no better data indicating otherwise. The following is a list of the searched keywords:

Invasion Adhesion Adhesin Invasion Outer Omp Outer Surface Porin Membrane Extensin Cell Surface Cell Wall Pilus Pilin Flagellar sheath BtuB Cir ChuA CopB ExeD FadL FecA FepA FhuA FmdC FomA FrpB GspD HemR HgbA Hgp HmbR HmuR HMW HrcC Hrp InvG LamB LbpA LcrQ Lmpl MxiD MOMP PilE HpaA NolW NspA OpcP OpnP Opr OspA PhoE PldA Por PscC Pu1D PupA QuiX RafY ScrY SepC ShuA SomA SpiA Tbpl Yop YscC mip Tol

Those ORFs that did not meet the minimum requirement for being an outer membrane protein based on the above search criteria but which were homologous to identified outer membrane ORFs in Chlamydia trachomatis were included. The Chlamydia trachomatis genome (French patent applications FR97-15041, filed Nov. 28, 1997 and FR97-16034 filed Dec. 17, 1997) was analyzed using the above search criteria and a number of outer membrane ORFs were identified. These Chlamydia trachomatis ORFs were then tested against the Chlamydia pneumoniae genome using Blastp. Any Chlamydia pneumoniae ORF with a Blastp P value better than e⁻¹⁰ against a Chlamydia trachomatis outer membrane was included in this section, if there was no better data indicating otherwise. A list of ORFs in the Chlamydia pneumoniae genome encoding putative surface exposed proteins is set forth above in the specification.

Identification of Putative Lipoproteins in the Genome of Chlamydia pneumoniae

Lipoproteins are the most abundant post-translationally modified bacterial secretory proteins (Pugsley, A. P. 1993. The complete general secretory pathway in Gram-negative bacteria. Microbiol. Rev. 57:50-108). The characteristic features of lipoproteins are a thiol-linked diacylglyceride and an amine-linked monoacyl group on the cysteine that becomes the amino-terminal residue after signal peptide cleavage by Signal Peptidase II. (Pugsley, A. P. 1993. The complete general secretory pathway in Gram-negative bacteria. Microbiol. Rev. 57:50-108). The identification of putative lipoproteins from the genomic sequencing of Chlamydia pneumoniae was done by examining the deduced amino acid sequence of identified ORFs for the presence of a signal peptide with a Signal Peptidase II cleavage site analogous to the consensus sequence for prolipoprotein modification and processing reactions (Hayashi, S., and H. C. Wu. 1992. Identification and characterization of lipid-modified proteins in bacteria, p. 261-285. In N. M. Hooper and A. J. Turner (ed.) Lipid modification of proteins: A practical approach. Oxford University Press, New York; Sutcliffe, I. C. and R. R. B. Russell. 1995. Lipoproteins of Gram-positive bacteria. J. Bacteriol. 177:1123-1128.).

Chlamydia pneumoniae ORFs were initially screened for the most basic of lipoprotein characteristics, a cysteine in the first 30 amino acids of the deduced protein. ORFs with a standard start codon (ATG, GTG, or TTG) and having one or more of the following characteristics were selected for direct analysis of their first 30 amino acids:

(a) Significant Signal P value (at least two out of the four values are Yes)

(b) PSORT value indicating membrane passage (IM-inner membrane, Peri-periplasm, or OM-outer membrane)

(c) Identification of the word lipoprotein among the ORF blastp data set.

(d) A Blastp value of <e⁻¹⁰ with a putative lipoprotein from Chlamydia trachomatis (French applications 97-15041 filed Nov. 28, 1997 and FR-97-16034 filed Dec. 17, 1997).

The first 30 amino acids of each ORF in this set were analyzed for the characteristics commonly found in lipoprotein signal peptides (Pugsley, A. P. 1993. The complete general secretory pathway in Gram-negative bacteria. Microbiol. Rev. 57:50-108; Hayashi, S., and H. C. Wu. 1992. Identification and characterization of lipid-modified proteins in bacteria, p. 261-285. In N. M. Hooper and A. J. Turner (ed.) Lipid modification of proteins: A practical approach. Oxford University Press, New York; Sutcliffe, I. C. and R. R. B. Russell. 1995. Lipoproteins of Gram-positive bacteria. J. Bacteriol. 177:1123-1128.) Putative lipoprotein signal peptides were required to have a cysteine between amino acid 10 and 30 and reach a minimum score of three based on the following criteria for lipoprotein signal peptides:

(a) Identification of specific amino acids in specific positions around the cysteine which are part of the consensus Signal Peptidase II cleavage site (Hayashi, S., and H. C. Wu. 1992. Identification and characterization of lipid-modified proteins in bacteria, p. 261-285. In N. M. Hooper and A. J. Turner (ed.) Lipid modification of proteins: A practical approach. Oxford University Press, New York); Sutcliffe, I. C. and R. R. B. Russell. 1995. Lipoproteins of Gram-positive bacteria. J. Bacteriol. 177:1123-1128). Since the identification of the cleavage site is the most important factor in identifying putative lipoproteins, each correctly positioned amino acid contributed toward reaching the minimum score of three.

(b) A hydrophobic region rich in alanine and leucine prior to the cleavage site (Pugsley, A. P. 1993. The complete general secretory pathway in Gram-negative bacteria. Microbiol. Rev. 57:50-108) contributed toward reaching the minimum score of three.

(c) A short stretch of hydrophilic amino acids greater than or equal to 1 usually lysine or arginine following the N-terminal methionine (Pugsley, A. P. 1993. The complete general secretory pathway in Gram-negative bacteria. Microbiol. Rev. 57:50-108) contributed toward reaching the minimum score of three.

A list of ORFs in the Chlamydia pneumoniae genome encoding putative lipoproteins is set forth above in the specification.

LPS-Related ORFs of Chlamydia pneumoniae

Lipopolysaccharide (LPS) is an important major surface antigen of Chlamydia cells. Monoclonal antibodies (Mab) directed against LPS of Chlamydia pneumoniae have been identified that can neutralize the infectivity of Chlamydia pneumoniae both in vitro and in vivo (Peterson, E. M., de la Maza, L. M., Brade, L., Brade, H. 1998. Characterization of a Neutralizing Monoclonal Antibody Directed at the Lipopolysaccharide of Chlamydia pneumonia. Infect. Immun. August 66(8):3848-3855.) Chlamydial LPS is composed of lipid A and a core oligosaccharide portion and is phenotypically of the rough type (R-LPS) (Lukacova, M., Baumann, M., Brade, L., Mamat, U., Brade, H. 1994. Lipopolysaccharide Smooth-Rough Phase Variation in Bacteria of the Genus Chlamydia. Infect. Immun. June 62(6):2270-2276.) The lipid A component is composed of fatty acids which serve to anchor LPS in the outer membrane. The core component contains sugars and sugar derivatives such as a trisaccharide of 3-deoxy-D-manno-octulosonic acid (KDO) (Reeves, P. R., Hobbs, M., Valvano, M. A., Skurnik, M., Whitfield, C., Coplin, D., Kido, N., Klena, J., Maskell, D., Raetz, C. R. H., Rick, P. D. 1996. Bacterial Polysaccharide Synthesis and Gene Nomenclature pp. 10071-10078, Elsevier Science Ltd.). The KDO gene product is a multifunctional glycosyltransferase and represents a shared epitope among the Chlamydia. For a review of LPS biosynthesis see, e.g., Schnaitman, C. A., Klena, J. D. 1993. Genetics of Lipopolysaccharide Biosynthesis in Enteric Bacteria. Microbiol. Rev. 57:655-682.

A text search of the ORF blastp results identified several genes that are involved in Chlamydial LPS production with a P score better than e⁻¹⁰. The following key-terms were used in the text search: KDO, CPS (Capsular Polysaccharide Biosynthesis), capsule, LPS, rfa, rfb, rfc, rfe, rha, rhl, core, epimerase, isomerase, transferase, pyrophosphorylase, phosphatase, aldolase, heptose, manno, glucose, lpxB, fibronectin, fibrinogen, fucosyltransferase , lic, lgt, pgm, tolC, rol, ChoP, phosphorylcholine, waaF, PGL-Tb1. A list of ORFs in the Chlamydia pneumoniae genome encoding putative polypeptides involved in LPS biosynthesis is set forth above in the specification.

Type III And Other Secreted Products

Type III secretion enables gram-negative bacteria to secrete and inject pathogenicity proteins into the cytosol of eukaryotic host cells (Hueck, C. J., 1998. Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants. In Microbiology and Molecular Biology Reviews. 62:379-433.) These secreted factors often resemble eukaryotic signal transduction factors, thus enabling the bacterium to redirect host cell functions (Lee, C. A., 1997. Type III secretion systems: machines to deliver bacterial proteins into eukaryotic cells? Trends Microbiol. 5:148-156.) In an attempt to corrupt normal cellular functions, Chlamydial pathogenicity factors injected into the host cytosol will nonetheless, as cytoplasmic constituents be processed and presented in the context of the Major Histocompatibility Complex (MHC class I). As such, these pathogenicity proteins represent MHC class I antigens and will play an important role in cellular immunity. Also included in this set are secreted non-type III products that may play a role as vaccine components.

A text search of the ORF blastp results identified genes that are involved in Chlamydia pneumoniae protein secretion with a P score better than e⁻¹⁰. The following key-terms were used in the text search in an effort to identify surface localized or secreted products: Yop, Lcr, Ypk, Exo, Pcr, Pop, Ipa, Vir, Ssp, Spt, Esp, Tir, Hrp, Mxi, hemolysin, toxin, IgA protease, cytolysin, tox, hap, secreted and Mip.

Chlamydia pneumoniae ORFs that did not meet the above keyword search criteria, but have homologs in Chlamydia trachomatis that do meet the search criteria are included herein. The Chlamydia trachomatis genome (French patent applications FR97-15041, filed Nov. 28, 1997 and 97-16034 filed Dec. 17, 1997) was analyzed using the above search criteria and a number of ORFs were identified. These Chlamydia trachomatis ORFs were tested against the Chlamydia pneumoniae genome using Blastp. Any Chlamydia pneumoniae ORF with a Blastp P value <e⁻¹⁰ against a Chlamydia trachomatis homolog, identified using the above search criteria, was included. A list of ORFs in the Chlamydia pneumoniae genome encoding putative secreted proteins is in the specification.

Chlamydia pneumoniae: RGD Recognition Sequence

Proteins that contain Arg-Gly-Asp (RGD) attachment site, together with integrins that serve as their receptor constitute a major recognition system for cell adhesion. The RGD sequence is the cell attachment site of a large number of adhesive extracellular matrix, blood, and cell surface proteins and nearly half of the known integrins recognize this sequence in their adhesion protein ligands. There are many RGD containing microbial proteins such as the penton protein of adenovirus, the coxsackie virus, the foot and mouth virus and pertactin, a 69 kDa (kilodalton) surface protein of Bordetella pertussis, that serve as ligands through which these microbes bind to integrins on the cell surfaces and gain entry into the cell. The following provides evidence supporting the importance of RGD in microbial adhesion:

a) The adenovirus penton base protein has a cell rounding activity and when penton base was expressed in E. coli, it caused cell rounding and cells adhered to polystyrene wells coated with the protein. Mutant analysis showed that both these properties required an RGD sequence. Virus mutants with amino acid substitutions in the RGD sequence, showed much less adherence to HeLa S3 cells, and also were delayed in virus reproduction (Bai, M., Harfe, B., and Freimuth, P. 1993. Mutations That Alter an RGD Sequence in the Adenovirus Type 2 Penton Base Protein Abolish Its Cell-Rounding Activity and Delay Virus Reproduction in Flat Cells. J. Virol. 67:5198-5205).

b) It has been shown that attachment and entry of coxsackie virus A9 to GMK cells were dependent on an RGD motif in the capsid protein VP1. VP1 has also been shown to bind a_(v)b₃ integrin, which is a vitronectin receptor (Roivainen, M., Piirainen, L., Hovi, T., Virtanen, I., Riikonen, T., Heino, J., and Hyypia, T. 1994. Entry of Coxsackievirus A9 into Host Cells: Specific Interactions with a_(v)b₃ Integrin, the Vitronectin Receptor Virology, 203:357-65).

c) During the course of whooping cough, Bordetella pertussis interacts with alveolar macrophages and other leukocytes on the respiratory epithelium. Whole bacteria adheres by means of two proteins, filamentous hemagglutinin (FHA) and pertussis toxin. FHA interacts with two classes of molecules on macrophages, galactose containing glycoconjugates and the integrin CR3. The interaction between CR3 and FHA involves recognition of RGD sequence at the positions 1097-1099 in FHA (Relman, D., Tuomanen, E., Falkow, S., Golenbock, D. T., Saukkonen, K., and Wright, S. D. “Recognitition of a Bacterial Adhesin by an Integrin: Macrophage CR3 Binds Filamentous Hemagglutinin of Bordetella Pertussis.” Cell, 61:1375-1382 (1990)).

d) Pertactin, a 69 kDa outer membrane protein of Bordetella pertussis, has been shown to promote attachment of Chinese hamster ovary cells (CHO). This attachment is mediated by recognition of RGD sequence in pertactin by integrins on CHO cells and can be inhibited by synthetic RGD containing peptide homologous to the one present in pertactin (Leininger, E., Roberts, M., Kenimer, J. G., Charles, I. G., Fairweather, N., Novotny, P., and Brennan, M. J. 1991. Pertactin, an Arg-Gly-Asp containing Bordetella pertussis surface protein that promotes adherence of mammalian cells Proc. Natl. Acad. Sci. USA, 88:345-349).

e) The RGD sequence is highly conserved in the VP1 protein of foot and mouth disease virus (FMDV). Attachment of FMDV to baby hamster kidney cells (BHK) has been shown to be mediated by VP1 protein via the RGD sequence. Antibodies against the RGD sequence of VP1 blocked attachment of virus to BHK cells (Fox, G., Parry, N. R., Barnett, P. V., McGinn, B., Rowland, D. J., and Brown, F. 1989. The Cell Attachment Site on Foot-and-Mouth Disease Virus Includes the Amino Acid Sequence RGD (Arginine-Glycine-Aspartic Acid) J. Gen. Virol., 70:625-637).

It has been demonstrated that bacterial adherence can be based on interaction of a bacterial adhesin RGD sequence with an integrin and that bacterial adhesins can have multiple binding site characteristic of eukaryotic extracellular matrix proteins. RGD recognition is one of the important mechanisms used by microbes to gain entry into eukaryotic cells.

The complete deduced protein sequence of the Chlamydia pneumoniae genome was searched for the presence of RGD sequence. There were a total of 54 ORFs that had one or more RGD sequences. Not all RGD containing proteins mediate cell attachment. It has been shown that RGD containing peptides that have proline immediately following the RGD sequence are inactive in cell attachment assays (Pierschbacher & Ruoslahti. 1987. Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion. J. Biol. Chem. 262:17294-98). ORFs that had RGD, with proline as the amino acid following the RGD sequence were excluded from the list. Also, RGD sequence may not be available at the surface of the protein or may be present in a context that is not compatible with integrin binding. Since not all RGD-containing proteins are involved in cell attachment, several other criteria were used to refine the list of RGD-containing proteins. A list of ORFs in the Chlamydia pneumoniae genome encoding polypeptides with RGD recognition sequence(s) is in the specification.

Non-Chlamydia trachomatis ORFs

Chlamydia pneumoniae ORFs were compared to the ORFs in the Chlamydia trachomatis genome (French patent applications FR97-15041, filed Nov. 28, 1997 and 97-16034 filed Dec. 17, 1997) using Blastp. Any Chlamydia pneumoniae ORF with a Blastp P value worse than e⁻¹⁰ (i.e. >e⁻¹⁰) against Chlamydia trachomatis ORFs are included in this section. A list of ORFs in the Chlamydia pneumoniae genome which are not found in Chlamydia trachomatis is set forth above in the specification.

Cell Wall Anchor Surface ORFs

Many surface proteins are anchored to the cell wall of Gram-positive bacteria via the conserved LPXTG motif (Schneewind, O., Fowler, A., and Faull, K. F. 1995. Structure of the Cell Wall Anchor of Surface Proteins in Staphylococcus aureus. Science 268:103-106). A search of the Chlamydia pneumoniae ORFs was done using the motif LPXTG. A list of ORFs in the Chlamydia pneumoniae genome encoding polypeptides anchored to the cell wall is in the specification.

ATCC Deposits

Samples of Chlamydia pneumoniae were deposited with the American Type Culture Collection (ATCC), Rockville, Md., on Nov. 19, 1998, and assigned the accession number VR-2634. Cells can be grown, harvested and purified, and DNA can be prepared as discussed above. In order to enable recovery of specific fragments of the chromosome, one can run targeted PCR reactions, whose amplification products can then be sequenced and/or cloned into any suitable vector, according to standard procedures known to those skilled in the art.

In addition, a sample of three pools of clones covering chromosomal regions of interest were deposited with the American Type Culture Collection (ATCC), Rockville, Md., on Nov. 19, 1998, and assigned the indicated accession numbers: 207000, 207001, and 207002. Each pool of clones contains a series of clones. When taken together, the three pools in the sample cover a portion of the chromosome, with a redundancy of slightly more than two. The total number of clones in the sample is 196.

The clones cover the following three regions of interest:

(i) position 30,000 to 40,000 of SEQ ID No. 1, referred to as region A;

(ii) position 501,500 to 557,000 of SEQ ID No. 1, referred to as region B; and

(iii) position 815,000 to 830,000 of SEQ ID No. 1, referred to as region C.

Table 4 lists groups of oligonucleotides to be used to amplify each of ORFs 2-1291 according to standard procedures known to those skilled in the art. Such oligonucleotides are listed as SEQ ID Nos. 1292 to 6451. For each ORF, the following is listed: one forward primer positioned 2,000 bp upstream of the beginning of the ORF; one forward primer positioned 200 bp upstream of the beginning of the ORF; one reverse primer positioned 2,000 bp downstream at the end of ORF, which is 2,000 bp upstream of the end site of the ORF on the complementary strand; and one reverse primer 200 bp downstream at the end of ORF, which is 200 bp upstream of the end site of the ORF on the complementary strand. The corresponding SEQ ID Nos. for the primers are listed in Table 4, where Fp is the proximal forward primer; Fd is the distal forward primer; Bp is the proximal reverse primer; and Bd is the distal reverse primer. The positions of the 5′ ends of each of these primers on the nucleotide sequence of SEQ ID No. 1 are shown in Table 5 (F refers to forward primers; R refers to reverse primers).

Table 6 lists oligonucleotides (SEQ ID Nos. 6452-6843) to be used to amplify the inserts of each of the 196 clones present in the pooled sample according to standard procedures well known to those of skill in the art. These primers can also be utilized to amplify the chromosomal region corresponding to the region A, B or C within which the particular insert lies. Their positions are indicated in Table 7 (F refers to forward primers; R refers to reverse primary).

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

TABLE 1 ORF Begin End Homology ID Species Score I % ORF2 42 794 triosephosphate isomerase L27492 Thermotoga maritima 567 54 ORF3 1258 1614 putative ORF4 1807 2418 polypeptide deformylase D90906 Synechocystis sp. 316 40 ORF5 3393 2491 hypothetical protein Z75208 Bacillus subtilis 338 42 ORF6 3639 4067 unknown U87792 Bacillus subtilis 117 38 ORF7 5649 4270 putative ORF8 7463 6012 putative ORF9 8051 8962 putative ORF10 9129 9959 putative ORF11 10687 10361 putative ORF12 10927 11232 putative ORF13 11246 12727 amidase U49269 Moraxella catarrhalis 1108 42 ORF14 12691 14190 PET112 D90913 Synechocystis sp. 1044 46 ORF15 14484 17249 POMP91A U65942 Chlamydia psittaci 1074 43 ORF16 16039 15770 putative ORF17 17845 20853 putative ORF18 21137 22042 putative ORF19 22046 23476 putative ORF20 23681 26110 putative ORF21 26109 25861 putative ORF22 26241 26978 putative ORF23 26960 27754 putative ORF24 27747 28577 putative ORF25 28887 29492 POMP91A U65942 Chlamydia psittaci 180 39 ORF26 29432 30028 POMP91A U65942 Chlamydia psittaci 361 51 ORF27 30024 31472 POMP91A U65942 Chlamydia psittaci 879 54 ORF28 31758 32288 putative 98 kDa outer membrane U72499 Chlamydia psittaci 144 43 protein ORF29 32201 33991 putative 98 kDa outer membrane U72499 Chlamydia psittaci 1126 48 protein ORF30 33852 34541 putative 98 kDa outer membrane U72499 Chlamydia psittaci 589 62 protein ORF31 34783 36063 POMP91B precursor U65943 Chlamydia psittaci 469 46 ORF32 36009 37529 putative 98 kDa outer membrane U72499 Chlamydia psittaci 1338 51 protein ORF33 37881 39362 putative 98 kDa outer membrane U72499 Chlamydia psittaci 671 40 protein ORF34 39418 39161 putative ORF35 39366 40715 POMP90A precursor U65942 Chlamydia psittaci 904 47 ORF36 43076 41094 putative ORF37 43800 43066 putative ORF38 44828 43785 putative ORF39 45340 44753 homologous to unidentified E. coli M96343 Bacillus subtilis 136 44 protein ORF40 45752 45372 o530; This 530 aa orf is 33 pct AE000184 Escherichia coli 269 43 identical (14 gaps) to 525 residues of an approx. 640 aa protein YHES_HAEIN SW: P44808 ORF41 46996 45701 ABC transporter, ATP-binding AE000596 Helicobacter pylori 878 39 protein (yheS) ORF42 47961 47569 putative ORF43 48960 48040 hypothetical protein D64001 Synechocystis sp. 404 37 ORF44 51452 50133 Lon protease-like protein X74215 Homo sapiens 1232 54 ORF45 52606 51335 unknown Z54285 Schizosaccharomyces pombe 781 47 ORF46 53684 53319 putative ORF47 54195 53746 putative ORF48 55278 56453 heat-shock protein U15010 Legionella pneumophila 975 45 ORF49 56493 57266 branched chain alpha-keto acid M97391 Bacillus subtilis 329 36 dehydrogenase E1-alpha ORF50 57297 58526 branched chain alpha-keto acid M97391 Bacillus subtilis 707 50 dehydrogenase E1-beta ORF51 59851 58565 putative ORF52 61495 59924 ComE D90903 Synechocystis sp. 134 55 ORF53 61324 62151 putative ORF54 62132 62470 Hpr protein X12832 Bacillus subtilis 136 36 ORF55 62474 63733 enzyme I (ptsI) U32844 Haemophilus influenzae 381 35 ORF56 63881 64186 f831; This 831 aa orf is 46 pct AE000326 Escherichia coli 123 34 identical (11 gaps) to 709 residues of an approx. 712 aa protein PT1A_ECOLI SW: P32670 ORF57 64611 64318 ORF107 X17014 Bacillus subtilis 128 33 ORF58 65485 64673 putative ORF59 65999 65301 dnaZX-like ORF put. DNA X06803 Bacillus subtilis 596 52 polymerase III ORF60 66244 67281 putative ORF61 67265 67699 putative ORF62 67703 68539 putative ORF63 68805 70736 putative ORF64 69172 68831 putative ORF65 70642 71142 putative ORF66 71325 72029 putative ORF67 72060 73637 putative ORF68 74061 76175 YqfF D84432 Bacillus subtilis 542 44 ORF69 78351 77680 porphobilinogen deaminase D28503 Clostridium josui 262 42 ORF70 79356 78355 sms protein D90914 Synechocystis sp. 736 52 ORF71 79983 79693 ribonuclease III (rnc) AE000579 Helicobacter pylori 98 33 ORF72 80441 79938 ORF3 D64116 Bacillus subtilis 268 44 ORF73 80475 80969 putative ORF74 81296 83080 hypothetical protein Y14079 Bacillus subtilis 893 38 ORF75 83291 83932 manganese superoxide dismutase X77021 Caenorhabditis elegans 622 58 ORF76 84005 84769 acetyl-CoA carboxylase beta subunit AE000604 Helicobacter pylori 602 50 (accD) ORF77 84975 85244 deoxyuridinetriphosphatase (dut) U32776 Haemophilus influenzae 110 41 ORF78 85123 85425 deoxyuridine 5′-triphosphate AE000596 Helicobacter pylori 265 68 nucleotidohydrolase (dut) ORF79 85397 85903 ORF2 L26916 Pseudomonas aeruginosa 173 34 ORF80 85909 86583 enzyme IIANtr U18997 Escherichia coli 170 42 ORF81 86626 88065 putative ORF82 89257 91026 putative ORF83 91291 93030 putative ORF84 93295 94086 putative ORF85 95285 94707 putative ORF86 95667 96557 putative ORF87 96317 97456 putative ORF88 98435 97968 putative ORF89 99460 98426 putative ORF90 100144 101325 elongation factor Tu L22216 Chlamydia trachomatis 1917 95 ORF91 101457 101720 putative ORF92 101704 102273 transcription factor L10348 Thermus aquaticus 376 49 thermophilus ORF93 102356 102805 ribosomal protein L11 D13303 Bacillus subtilis 458 63 ORF94 102835 103530 ribosomal protein L1 Z11839 Thermotoga maritima 642 51 ORF95 103549 104058 ribosomal protein L10 M89911 Streptomyces antibioticus 82 31 ORF96 104096 104491 rp112 (AA 1-128) X53178 Synechocystis PCC6803 325 47 ORF97 104601 108386 DNA-directed RNA polymerase beta X64172 Staphylococcus aureus 2740 52 chain ORF98 108401 112054 rpoC V00339 Escherichia coli 2947 54 ORF99 112033 112590 acetylornithine deacetylase (EC M22622 Leptospira biflexa 514 62 5.1.1.16) ORF100 112672 113682 transaldolase L19437 Homo sapiens 755 49 ORF101 113726 114121 putative ORF102 114711 114136 putative ORF103 115267 115755 putative ORF104 115911 116543 putative ORF105 116736 118055 ATPase alpha-subunit X63855 Thermus aquaticus 934 50 thermophilus ORF106 117968 118522 adenosine triphosphatase A subunit D50528 Acetabularia acetabulum 147 32 ORF107 118530 119843 V-ATPase B subunit U96487 Desulfurococcus sp. SY 751 48 ORF108 119816 120457 putative ORF109 120451 122430 v-type Na-ATPase X76913 Enterococcus hirae 264 35 ORF110 122504 122950 ATP synthase, subunit K U67478 Methanococcus jannaschii 184 31 ORF111 123528 126347 valyl-tRNA synthetase X05891 Escherichia coli 1679 49 ORF112 126332 129166 protein kinase-like protein U19250 Streptomyces coelicolor 427 37 ORF113 134690 129213 UvrA D49911 Thermus thermophilus 3107 41 ORF114 134925 136382 pyruvate kinase U83196 Chlamydia trachomatis 1748 71 ORF115 137870 136482 HtrB protein X61000 Escherichia coli 147 38 ORF116 137899 138240 putative ORF117 138239 137928 putative ORF118 139558 138257 putative ORF119 140352 139516 YbbP AB002150 Bacillus subtilis 231 46 ORF120 140498 141841 cyanide insensitive terminal oxidase Y10528 Pseudomonas aeruginosa 538 50 ORF121 141855 142658 cyanide insensitive terminal oxidase Y10528 Pseudomonas aeruginosa 310 40 ORF122 144258 143050 putative ORF123 145258 144494 putative ORF124 145454 146749 product similar to E. coli PhoH Z97025 Bacillus subtilis 836 47 protein ORF125 147318 146767 putative ORF126 148261 147677 putative ORF127 149029 152157 isoleucyl-tRNA synthetase U04953 Homo sapiens 2361 52 ORF128 154108 152201 leader peptidase I D90904 Synechocystis sp. 225 47 ORF129 155135 154308 putative ORF130 155141 155467 YtiA AF008220 Bacillus subtilis 201 43 ORF131 155703 156779 orf 361; ranslated orf similarity to X78969 Coxiella burnetii 863 59 SW: RF1_SALTY peptide chain release factor 1 of Salmonella typhimurium ORF132 156748 157635 product similar to E. coli PRFA2 Z49782 Bacillus subtilis 144 37 protein ORF133 157653 158996 Ffh U82109 Thermus aquaticus 797 45 ORF134 159363 159986 tRNA U32705 Haemophilus influenzae 545 49 (guanine-N1)-methyltransferase (trmD) ORF135 159880 160446 putative ORF136 160477 160839 ribosomal protein L19 X72627 Synechocystis sp. 319 50 ORF137 160898 161539 putative protein highly homologous D32253 Magnetospirillum sp. 427 49 to E. coli RNase HII. ORF138 161527 162153 5′ guanylate kinase (gmk) U32848 Haemophilus influenzae 385 43 ORF139 162144 162443 putative ORF140 162437 164098 methionyl-tRNA synthetase AB004537 Schizosaccharomyces pombe 861 54 ORF141 165451 164228 exodeoxyribonuclease V (recD) U32811 Haemophilus influenzae 432 32 ORF142 166349 165411 putative ORF143 166949 168442 putative ORF144 169416 171029 putative ORF145 170857 171459 putative ORF146 172652 173428 putative biotin-protein ligase Z97992 Schizosaccharomyces pombe 292 44 ORF147 174626 173439 putative ORF148 174816 175613 putative ORF149 175598 175954 putative ORF150 175958 176935 putative ORF151 177708 176938 orf 3′ of chaperonin homolog hypB S40172 Chlamydia psittaci 376 74 [Chlamydia psittaci, pigeon strain P-1041, Peptide Partial, 98 aa] ORF152 177128 177376 putative ORF153 179472 177841 putative M69217 Chlamydia pneumoniae 2678 100 ORF154 179822 179517 putative M69217 Chlamydia pneumoniae 498 99 ORF155 181793 179943 Pz-peptidase D88209 Bacillus licheniformis 1088 38 ORF156 182628 181876 o247; This 247 aa orf is 51 pct AE000174 Escherichia coli 401 42 identical (0 gaps) to 117 residues of an approx. 160 aa protein YPH7_CHRVI SW: P45371 ORF157 184420 183074 glutamate-1-semialdehyde X53696 Escherichia coli 823 41 2,1-aminomutase ORF158 184988 184467 ORF_o211 U28377 Escherichia coli 87 54 ORF159 185483 185112 hypothetical protein D90906 Synechocystis sp. 91 33 ORF160 185902 185483 ribose 5-phosphate isomerase U28377 Escherichia coli 111 41 ORF161 186174 185839 ribose 5-phosphate isomerase A U32729 Haemophilus influenzae 190 46 (SP: P27252) ORF162 187720 186587 hypothetical D83026 Bacillus subtilis 536 42 ORF163 188318 190933 ATP-dependent protease binding M29364 Escherichia coli 2010 53 subunit ORF164 191090 191635 putative ORF165 191547 192743 putative ORF166 192969 193469 putative ORF167 194044 193610 putative ORF168 194196 195809 unknown Z84395 Mycobacterium tuberculosis 242 52 ORF169 196088 198073 DNA ligase (EC 6.5.1.2) M24278 Escherichia coli 1317 46 ORF170 198132 199454 putative ORF171 199351 202818 putative ORF172 204552 202999 PcpB U60175 Sphingomonas 80 41 chlorophenolica ORF173 205648 204692 putative ORF174 205807 207327 leucine tRNA synthetase AF008220 Bacillus subtilis 1595 57 ORF175 207182 207775 leucyl-tRNA synthetase X06331 Escherichia coli 363 51 ORF176 207779 208267 transfer RNA-Leu synthetase M88581 Bacillus subtilis 285 43 ORF177 208267 209577 KDO-transferase Z31593 Chlamydia pneumoniae 2262 100 ORF178 211807 211271 KDO-transferase X80061 Chlamydia psittaci 105 38 ORF179 212188 211844 putative ORF180 214079 212448 pyrophosphate-dependent Z32850 Ricinus communis 1003 45 phosphofructokinase beta subunit ORF181 214907 214083 CinI U44893 Butyrivibrio fibrisolvens 111 41 ORF182 216154 215429 putative ORF183 216115 216678 putative ORF184 216728 217282 putative ORF185 217267 217866 putative ORF186 218593 218261 putative ORF187 219821 218994 putative ORF188 221382 220309 putative ORF189 222719 221433 GMP synthetase M10101 Escherichia coli 1151 48 ORF190 223521 222724 IMP dehydrogenase X66859 Acinetobacter calcoaceticus 778 58 ORF191 224499 225008 putative ORF192 225140 225559 putative ORF193 225555 226802 putative ORF194 227800 226892 putative ORF195 228335 228072 putative ORF196 229251 228643 putative ORF197 230983 229622 YqhX D84432 Bacillus subtilis 1386 56 ORF198 231483 230983 acetyl-CoA carboxylase biotin U38804 Porphyra purpurea 199 52 carboxyl carrier protein ORF199 232063 231509 elongation factor P D64001 Synechocystis sp. 282 32 ORF200 232739 232053 pentose-5-phosphate-3-epimerase D90911 Synechocystis sp. 463 43 ORF201 233166 234356 putative ORF202 233518 233165 putative ORF203 234536 235186 ORF2 L35036 Chlamydia psittaci 570 60 ORF204 235379 236689 putative ORF205 236680 237618 putative ORF206 237521 238345 putative ORF207 238281 238973 putative ORF208 238871 240115 putative ORF209 240191 241564 putative ORF210 242281 241604 YqiZ D84432 Bacillus subtilis 379 39 ORF211 242933 242274 f222; This 222 aa orf is 48 pct AE000284 Escherichia coli 382 45 identical (0 gaps) to 208 residues of an approx. 232 aa protein YCKA_BACSU SW: P42399 ORF212 243416 242976 arginine repressor protein (argR) U32800 Haemophilus influenzae 229 46 ORF213 243500 244531 sialoglycoprotease U15958 Pasteurella haemolytica 565 53 ORF214 244480 246021 oligopeptide permease homolog AII AF000366 Borrelia burgdorferi 457 34 ORF215 246330 247811 OppAIV AF000948 Borrelia burgdorferi 453 35 ORF216 247831 249174 OppA gene product X56347 Bacillus subtilis 255 37 ORF217 249437 251038 dciAE X56678 Bacillus subtilis 469 37 ORF218 251325 252212 OppB gene product X56347 Bacillus subtilis 652 42 ORF219 253156 254007 oligopeptidepermease X89237 Streptococcus pyogenes 574 48 ORF220 253974 254852 ATP binding protein L18760 Lactococcus lactis 433 40 ORF221 255258 256094 KDO-transferase X80061 Chlamydia psiitaci 106 46 ORF222 256640 257455 putative ORF223 257502 258239 2-OXOGLUTARAT A47930 Spinacia oleracea 636 52 ORF224 257869 257501 putative ORF225 259248 260897 pyrophosphate-fructose 6-phosphate M55191 Solanum tuberosum 1055 44 1-phosphotransferase beta-subunit ORF226 262753 261788 putative ORF227 263059 262757 putative ORF228 264375 263182 putative ORF229 265985 264747 putative ORF230 266637 266059 putative ORF231 267338 266538 putative ORF232 267922 267473 putative ORF233 269647 270771 tRNA guanine transglycosylase L33777 Zymomonas mobilis 628 44 ORF234 272777 273145 ORF4 D00624 Bacteriophage chp1 100 41 ORF235 273253 273636 putative ORF236 273705 273977 putative ORF237 276016 275717 putative ORF238 276439 276020 putative ORF239 276792 277253 putative ORF240 277318 277599 putative ORF241 278578 277877 putative ORF242 279258 278554 FbpC U33937 Neisseria gonorrhoeae 312 39 ORF243 280435 279533 putative ORF244 281547 280849 putative ORF245 281696 282325 CMP-2-keto-3-deoxyoctulosonic U15192 Chlamydia trachomatis 637 63 acid synthetase ORF246 282459 284069 CTP synthetase U15192 Chlamydia trachomatis 2000 68 ORF247 284056 284517 ORF3 U15192 Chlamydia trachomatis 453 65 ORF248 284606 285775 glucose 6-phosphate dehydrogenase U83195 Chlamydia trachomatis 1263 77 ORF249 285592 285987 glucose 6-phosphate dehydrogenase U83195 Chlamydia trachomatis 519 79 ORF250 286179 286976 glucose-6-phosphate dehydrogenase D88189 Actinobacillus 216 40 isozyme actinomycetemcomitans ORF251 287583 287002 putative ORF252 287951 287451 putative ORF253 288499 288816 putative ORF254 289674 288505 putative ORF255 288839 289213 putative ORF256 289970 290254 putative ORF257 291931 292803 gamma-D-glutamyl-L-diamino acid X64809 Bacillus sphaericus 95 39 endopeptidase II ORF258 293258 292755 ScoS9 U43429 Streptomyces coelicolor 233 45 ORF259 293718 293272 ribosomal protein L13 (rpLl3) U32823 Haemophilus influenzae 364 47 ORF260 294630 293953 glutamine transport ATP-binding U67524 Methanococcus jannaschii 387 46 protein Q ORF261 296153 294636 putative ORF262 294817 295068 putative ORF263 296354 297862 conserved hypothetical protein AE000586 Helicobacter pylori 641 46 ORF264 298415 297879 putative ORF265 298777 298253 putative ORF266 299572 298781 putative ORF267 300487 299633 putative ORF268 301586 300702 putative ORF269 302440 301571 putative ORF270 302838 302437 putative ORF271 303335 302745 putative ORF272 304394 303852 putative ORF273 304606 305223 f311; This 311 aa orf is 22 pct AE000232 Escherichia coli 250 38 identical (13 gaps) to 186 residues of an approx. 488 aa protein YACA_BACSU SW: P37563; pyul of D21139 ORF274 305394 306236 survival protein surE U81296 Sinorhizobium meliloti 156 42 ORF275 306501 307439 YqfU D84432 Bacillus subtilis 547 42 ORF276 308033 307458 3-octaprenyl-4-hydroxybenzoate U61168 Bacillus firmus 403 42 carboxy-lyase ORF277 308924 308037 4-hydroxybenzoate U61168 Bacillus firmus 152 40 octaprenyltransferase ORF278 309485 310180 putative ORF279 310426 311214 putative ORF280 311597 311253 putative ORF281 312772 311780 putative ORF282 313425 312772 putative ORF283 313646 313377 putative ORF284 313937 314665 lysophospholipase homolog AF006678 Schistosoma mansoni 141 44 ORF285 315576 314755 dnaZX X17014 Bacillus subtilis 154 39 ORF286 316157 315531 unknown D26185 Bacillus subtilis 284 31 ORF287 318657 316156 DNA gyrase L47978 Aeromonas salmonicida 1785 48 ORF288 321042 318676 DNA gyrase subunit B U35453 Clostridium acetobutylicum 1838 59 ORF289 321445 321098 putative ORF290 322309 321710 putative ORF291 323190 322366 outer membrane protein AE000654 Helicobacter pylori 376 43 ORF292 323843 323181 hypothetical U70214 Escherichia coli 356 37 ORF293 324878 323856 ATP-binding protein (abc) U32744 Haemophilus influenzae 545 44 ORF294 325340 326410 f374; This 374 aa orf is 30 pct AE000299 Escherichia coli 1194 62 identical (9 gaps) to 102 residues of an approx. 512 aa protein FLIC_SALMU SW: P06177 ORF295 326433 327836 Xas A AE000246 Escherichia coli 479 33 ORF296 328465 327839 putative ORF297 329360 328857 putative ORF298 330907 329357 putative ORF299 332455 330956 MgtE U18744 Bacillus firmus 203 36 ORF300 334536 332395 putative ORF301 336091 334877 putative ORF302 336103 337302 putative ORF303 338129 338830 putative ORF304 338965 339501 putative ORF305 339508 340143 putative ORF306 340247 342967 putative ORF307 343385 343810 cAMP-dependent protein kinase type U75932 Rattus norvegicus 102 37 I regulatory subunit ORF308 344171 343935 acyl carrier protein (acpP) AE000570 Helicobacter pylori 198 55 ORF309 345082 344330 3-ketoacyl-ACP reductase U39441 Vibrio harveyi 598 48 ORF310 346005 345082 malonyl-CoA: Acyl carrier protein U59433 Bacillus subtilis 538 45 transacylase ORF311 346784 346437 beta-ketoacyl-acyl carrier protein AE000540 Helicobacter pylori 273 50 synthase III (fabH) ORF312 347029 346715 beta-ketoacyl-acyl carrier protein M77744 Escherichia coli 265 63 synthase III ORF313 347034 347723 recombination protein D90916 Synechocystis sp. 363 42 ORF314 348075 350459 putative ORF315 350598 351071 putative ORF316 351075 352175 rifampicin resistance protein L22690 Rickettsia rickettsii 495 46 ORF317 353291 352230 putative ORF318 353442 354467 pyruvate dehydrogenase E1 D90915 Synechocystis sp. 571 44 component, alpha subunit ORF319 354451 354933 pyruvate dehydrogenase E1 beta U09137 Arabidopsis thaliana 495 59 subunit ORF320 355000 355449 pyruvate dehydrogenase E1 U38804 Porphyra purpurea 336 47 component, beta subunit ORF321 355448 356743 F23B12.5 Z77659 Caenorhabditis elegans 759 46 ORF322 355953 355642 putative ORF323 359310 356827 glycogen phosphorylase B U47025 Homo sapiens 2193 57 ORF324 359120 359377 putative ORF325 359525 359908 putative ORF326 361290 359947 DnaA D89066 Staphylococcus aureus 375 46 ORF327 363785 361362 hypothetical U32781 Haemophilus influenzae 394 44 ORF328 364496 363888 putative ORF329 364832 365290 putative ORF330 365304 365669 dpj M76470 Escherichia coli 160 45 ORF331 366599 365667 NADPH thioredoxin reductase AC002329 Arabidopsis thaliana 975 60 ORF332 367291 369030 ribosomal protein S1 (rpS1) U32801 Haemophilus influenzae 1209 41 ORF333 369134 369808 NusA U74759 Chlamydia trachomatis 995 87 ORF334 369917 370438 NusA U74759 Chlamydia trachomatis 760 87 ORF335 370365 372647 U74759 Chlamydia trachomatis 2173 61 ORF336 372557 373066 initiation factor IF2-beta (infB; gtg X00513 Escherichia coli 333 39 start codon) ORF337 373020 373442 ORF6 gene product Z18631 Bacillus subtilis 192 34 ORF338 373467 374195 tRNA pseudouridine 55 synthase D90917 Synechocystis sp. 358 47 ORF339 374176 375099 hypothetical 34.6 kD protein in AE000113 Escherichia coli 395 39 rpsT-ileS intergenic region ORF340 375676 375083 hypothetical GTP-binding protein in AE000219 Escherichia coli 507 53 pth 3′ region ORF341 376173 375634 hypothetical U32723 Haemophilus influenzae 480 59 ORF342 376564 377643 YscU U08019 Yersinia enterocolitica 538 37 ORF343 377956 379773 IcrD gene product X67771 Yersinia enterocolitica 1302 47 ORF344 379781 380425 putative ORF345 380281 381000 putative ORF346 381008 381460 putative ORF347 381460 383037 4-alpha-glucanotransferase L37874 Clostridium butyricum 302 38 ORF348 383257 383523 ribosomal protein L28 (rpL28) U32776 Haemophilus influenzae 175 55 ORF349 383553 385304 hypothetical protein D90901 Synechocystis sp. 565 38 ORF350 385397 386458 comE ORF1 D64002 Synechocystis sp. 187 10 ORF351 387242 386514 putative ORF352 388764 387013 putative ORF353 390120 390932 methylenetetrahydrofolate D64000 Synechocystis sp. 588 53 dehydrogenase ORF354 390919 391818 f351; Residues 1-121 are 100 pct AE000310 Escherichia coli 186 39 identical to YOJL_ECOLI SW: P33944 (122 aa) and aa 152-351 are 100 pct identical to YOJK_ECOLI SW: P33943 ORF355 392379 391885 small protein D90914 Synechocystis sp. 387 46 ORF356 392582 392986 putative ORF357 392776 393684 putative ORF358 394151 394804 RecF protein D90907 Synechocystis sp. 232 34 ORF359 394928 395308 putative ORF360 395259 395990 putative ORF361 397815 395953 hypothetical U32773 Haemophilus influenzae 391 36 ORF362 398850 397831 H. influenzae predicted coding U32763 Haemophilus influenzae 580 39 region H10807 ORF363 400085 399099 putative ORF364 401245 400073 YtgC AF008220 Bacillus subtilis 244 30 ORF365 401474 401136 putative ORF366 402199 401423 unknown U52850 Erysipelothrix rhusiopathiae 534 46 ORF367 403193 402186 putative ORF368 403650 404165 putative ORF369 404343 405914 adenine nucleotide translocase Z49227 Arabidopsis thaliana 1280 55 ORF370 405984 407327 putative ORF371 407712 408806 putative ORF372 410439 409075 putative ORF373 411826 410954 putative ORF374 412482 414302 lepA gene product X91655 Bacillus subtilis 1827 59 ORF375 415402 414407 6-phosphogluconate dehydrogenase, U32737 Haemophilus influenzae 687 51 decarboxylating (gnd) ORF376 415848 415237 6-phosphogluconate dehydrogenase, S67873 Ceratitis capitata 695 64 6PGD [Ceratitis capitata = medflies, Peptide, 481 aa] ORF377 417131 415866 tyrosyl-tRNA synthetase (tyrS) J01719 Escherichia coli 821 45 ORF378 417258 417566 putative ORF379 418326 417454 whiG-Stv gene product X68709 Streptoverticillium 464 41 griseocarneum ORF380 420057 418426 FLHA gene product X63698 Bacillus subtilis 455 49 ORF381 420448 420720 ferredoxin IV M59855 Rhodobacter capsulatus 174 63 ORF382 420980 421552 putative ORF383 421556 422029 putative ORF384 422461 422925 putative ORF385 423562 424320 putative ORF386 424250 424591 putative ORF387 424830 426047 putative ORF388 426240 427397 putative ORF389 428841 430703 GcpE D90908 Synechocystis sp. 877 47 ORF390 430694 431446 YfiH U50134 Escherichia coli 136 35 ORF391 431597 432100 putative ORF392 432165 432779 putative ORF393 433272 432832 dihydrolipoamide U32839 Haemophilus influenzae 475 64 succinyltransferase (sucB) ORF394 433925 433227 dihydrolipoamide U32839 Haemophilus influenzae 332 45 succinyltransferase (sucB) ORF395 436678 433934 alpha-ketoglutarate dehydrogenase U41762 Rhodobacter capsulatus 1530 44 ORF396 437176 438357 oxygen-independent AE000628 Helicobacter pylori 442 42 coproporphyrinogen III oxidase (hemN) ORF397 440317 438518 putative ORF398 440001 440345 putative ORF399 441233 440517 ORF_f286 U18997 Escherichia coli 168 45 ORF400 440719 441012 putative ORF401 442192 441230 putative ORF402 442888 442343 putative ORF403 442371 442961 putative ORF404 443578 443003 [karp] gene products M86605 Chlamydia trachomatis 505 78 ORF405 444500 443526 aminopeptidase D17450 Mycoplasma salivarium 273 39 ORF406 444842 444528 putative ORF407 445009 444743 putative L39923 Mycobacterium leprae 133 33 ORF408 445718 445182 putative ORF409 445807 447804 Su1p U18908 Zea mays 1307 52 ORF410 448738 447803 putative ORF411 449628 448618 RuvB protein U38840 Thermotoga maritima 845 53 ORF412 450298 450867 deoxycytidine triphosphate AE000554 Helicobacter pylori 573 58 deaminase (dcd) ORF413 450713 451207 putative ORF414 451211 452452 hemolysin D90914 Synechocystis sp. 227 39 ORF415 452448 453659 similar to [SwissProt Accession D90888 Escherichia coli 96 33 Number P37908] ORF416 454843 453725 NifS gene product L34879 Anabaena azollae 533 38 ORF417 455608 454865 hypothetical protein D90908 Synechocysus sp. 371 36 ORF418 456243 457007 putative ORF419 457016 457708 putative ORF420 458368 457979 unknown D26185 Bacillus subtilis 152 36 ORF421 459496 458372 mutY homolog U63329 Homo sapiens 466 46 ORF422 459493 460194 hypothetical protein D90914 Synechocystis sp. 98 38 ORF423 461446 460355 putative ORF424 462298 461450 putative ORF425 462444 463349 enoyl-ACP reductase Y13861 Nicotiana tabacum 1008 69 ORF426 464241 463342 putative ORF427 464574 465065 putative ORF428 465129 465611 putative ORF429 465571 466317 putative ORF430 466317 467093 H. pylori predicted coding region AE000536 Helicobacter pylori 246 36 HP0152 ORF431 466999 467502 putative ORF432 469691 467715 unidentified transporter-ATP binding Z82044 Bacillus subtilis 496 45 ORF433 470691 469660 acetyl-CoA carboxylase subunit AF008220 Bacillus subtilis 781 52 ORF434 472010 470709 putative ORF435 471545 471799 putative ORF436 472359 472045 putative ORF437 473523 472732 orf1 X75413 Escherichia coli 313 42 ORF438 474889 473441 murE gene product Z15056 Bacillus subtilis 679 37 ORF439 477323 475365 penicillin-binding protein 2 X59630 Neisseria meningitidis 451 42 ORF440 478496 477597 hypothetical protein D90906 Synechocystis sp. 534 52 ORF441 478722 479273 putative ORF442 479277 479705 putative ORF443 480050 481450 chromosomal replication initiator D90909 Synechocystis sp. 793 40 protein DnaA ORF444 481469 482053 OrfH U35673 Borrelia burgdorferi 157 37 ORF445 482600 482025 putative ORF446 482654 484204 NADH: ubiquinone oxidoreductase Z37111 Vibrio alginolyticus 801 49 subunit B ORF447 484211 485170 NADH: ubiquinone oxidoreductase U32702 Haemophilus influenzae 258 48 (GP: Z37111_4) ORF448 485170 485838 NADH: uniquinone oxidoreductase Z37111 Vibrio alginolyticus 543 55 ORF449 485813 486580 unidentified protein of D49364 Vibrio alginolyticus 488 48 Na+-translocating NADH-quinone reductase ORF450 486976 486638 putative ORF451 489071 487764 putative ORF452 489341 489090 putative ORF453 489958 489152 putative ORF454 490549 489962 putative ORF455 491163 490522 putative ORF456 491396 491112 putative ORF457 492121 491390 putative ORF458 492304 494838 ClpC adenosine triphosphatase U02604 Bacillus subtilis 2370 46 ORF459 495943 494822 hypothetical protein in purB 5′ AE000213 Escherichia coli 927 53 region ORF460 496011 496565 putative ORF461 496569 497228 putative ORF462 497358 497834 putative ORF463 497770 498327 putative ORF464 499209 499589 putative ORF465 499520 499792 putative ORF466 500774 504169 putative 98 kDa outer membrane U72499 Chlamydia psittaci 1215 45 protein ORF467 504139 504600 putative 98 kDa outer membrane U72499 Chlamydia psittaci 319 47 protein ORF468 504865 506877 putative 98 kDa outer membrane U72499 Chlamydia psittaci 992 42 protein ORF469 506790 507671 putative 98 kDa outer membrane U72499 Chlamydia psittaci 739 46 protein ORF470 507718 510507 putative 98 kDa outer membrane U72499 Chlamydia psittaci 1813 42 protein ORF471 508325 507912 putative ORF472 510660 513440 POMP90A precursor U65942 Chlamydia psittaci 1830 46 ORF473 514965 513787 hypothetical D83026 Bacillus subtilis 482 48 ORF474 517347 515419 putative 98 kDa outer membrane U72499 Chlamydia psittaci 1554 51 protein ORF475 517058 517363 putative ORF476 517798 517277 putative 98 kDa outer membrane U72499 Chlamydia psittaci 222 41 protein ORF477 518200 517847 POMP91B precursor U65943 Chlamydia psittaci 162 42 ORF478 518300 521146 putative 98 kDa outer membrane U72499 Chlamydia psittaci 1900 45 protein ORF479 521392 522948 POMP91A U65942 Chlamydia psittaci 490 39 ORF480 523244 524809 putative 98 kDa outer membrane U72499 Chlamydia psittaci 507 35 protein ORF481 524379 524125 putative ORF482 524649 526238 putative 98 kDa outer membrane U72499 Chlamydia psittaci 969 41 protein ORF483 526265 527104 putative ORF484 526947 526702 putative ORF485 526975 528450 putative 98 kDa outer membrane U72499 Chlamydia psittaci 197 48 protein ORF486 528408 529199 putative outer membrane protein U72499 Chlamydia psittaci 154 37 ORF487 530612 529542 putative ORF488 531656 530616 putative ORF489 533974 532067 putative ORF490 536432 534324 putative ORF491 537150 536707 putative ORF492 537928 537080 putative ORF493 538438 537932 putative ORF494 538737 538333 putative ORF495 539594 539127 putative ORF496 541215 539590 putative ORF497 542571 541282 putative ORF498 543014 542457 putative ORF499 543369 542962 putative ORF500 543809 546628 putative 98 kDa outer membrane U72499 Chlamydia psittaci 506 89 protein ORF501 546619 549525 POMP91A U65942 Chlamydia psittaci 128 50 ORF502 547293 546994 putative ORF503 549699 550523 putative 98 kDa outer membrane U72499 Chlamydia psittaci 96 32 protein ORF504 550490 551551 putative 98 kDa outer membrane U72499 Chlamydia psittaci 223 33 protein ORF505 551448 552623 putative 98 kDa outer membrane U72499 Chlamydia psittaci 139 46 protein ORF506 552652 555117 putative 98 kDa outer membrane U72499 Chlamydia psittaci 487 48 protein ORF507 555029 555493 putative ORF508 558006 555673 putative ORF509 559694 558162 putative ORF510 558208 558573 putative ORF511 561692 559899 putative ORF512 561412 561708 putative ORF513 563942 561777 1,4-alpha-glucan branching enzyme X73903 Streptomyces coelicolor 1743 45 ORF514 564969 563950 putative ORF515 566204 564936 YqeV D84432 Bacillus subtilis 639 38 ORF516 567717 566302 putative GTPase required for high U00005 Escherichia coli 686 41 frequency lysogenization by bacteriophage lambda ORF517 568526 567708 putative ORF518 569467 568742 putative ORF519 571065 569431 putative ORF520 571828 571118 arginine-binding periplasmic protein AE000188 Escherichia coli 197 45 1 precursor ORF521 572202 573308 putative ORF522 573146 575056 putative ORF523 575023 575916 carboxysome formation protein D90901 Synechocystis sp. 557 59 ORF524 577891 576497 putative ORF525 578914 578204 putative ORF526 579924 578857 putative ORF527 580187 579858 protein kinase C inhibitor D90906 Synechocystis sp. 260 49 ORF528 580017 580406 putative ORF529 581086 580187 Yer156cp U18917 Saccharomyces cerevisiae 176 34 ORF530 581367 581828 putative ORF531 581678 582367 putative ORF532 582361 583428 putative ORF533 584690 583431 putative ORF534 585237 584950 putative ORF535 585626 586888 hypothetical protein D64004 Synechocystis sp. 805 45 ORF536 586846 587907 putative ORF537 589049 588180 putative ORF538 590500 589301 putative ORF539 590755 592458 aminoacyl-tRNA synthetase L25105 Chlamydia trachomatis 2125 71 ORF540 592526 592903 has homology to putative heat shock L25105 Chlamydia trachomatis 324 59 proteins of Bacillus subtilis and Clostridium acetobutylicum; ORFA; putative ORF541 592836 593747 Possible negative regulator of U52216 Chlamydia trachomatis 960 65 CIRCE element; Homologs in B. subtilis and Clostridia spp. referred to as hrcA or orfA ORF542 593747 594298 grpE M62819 Chlamydia trachomatis 661 71 ORF543 594331 595947 DnaK protein homolog; 71,550 Da; M69227 Chlamydia pneumoniae 2619 100 putative ORF544 595905 596309 DnaK protein homolog; 71,550 Da; M69227 Chlamydia pneumoniae 674 100 putative ORF545 596514 597215 putative ORF546 597184 597957 vacB gene product U14003 Escherichia coli 306 48 ORF547 597755 598612 ORF-2 D11024 Shigella flexneri 168 46 ORF548 598602 599204 homologous to DNA glycosylases; D83026 Bacillus subtilis 374 47 hypothetical ORF549 599373 599939 putative ORF550 600903 602072 hemolysin X73141 Serpulina hyodysenteriae 362 36 ORF551 602240 602587 hypothetical protein D90908 Synechocystis sp. 182 35 ORF552 602637 603272 putative ORF553 603142 604512 putative ORF554 604627 605853 conserved hypothetical protein AE000579 Helicobacter pylori 423 40 ORF555 605790 606620 putative ORF556 606571 607281 putative L14679 Lactococcus lactis 384 45 ORF557 609004 607355 putative ORF558 610906 609932 putative ORF559 611786 611004 diaminopimelate epimerase D90917 Synechocystis sp. 207 55 ORF560 612333 611746 ATP-dependent Clp protease D90915 Synechocystis sp. 389 44 proteolytic subunit ORF561 613897 612341 serine hydroxymethyltransferase D90903 Synechocystis sp. 909 52 ORF562 615179 616279 putative ORF563 616610 617383 putative ORF564 618796 617810 ORF_o328 U18997 Esherichia coli 413 45 ORF565 620004 618826 branched chain alpha-keto acid M97391 Bacillus subtilis 688 41 dehydrogenase E2 ORF566 619649 619918 putative ORF567 621265 620021 Hypothetical protein Y14083 Bacillus subtilis 727 37 ORF568 622359 621265 hypothetical U32691 Haemophilus influenzae 294 52 ORF569 623420 622560 rRNA methylase D90913 Synechocyslis sp. 244 38 ORF570 624297 623335 hypothetical protein (SP: P39587) U67605 Methanococcus jannaschii 147 35 ORF571 624773 624174 riboflavin synthase alpha chain AE000261 Escherichia coli 424 50 ORF572 625029 625484 ORF168 D28752 Synechococcus sp. 323 43 ORF573 625488 625883 YteA AF008220 Bacillus subtilis 172 35 ORF574 625892 626395 signalpeptidase II X78084 Staphylococcus carnosus 204 38 ORF575 626444 627790 D-alanine permease (dagA) U32770 Haemophilus influenzae 566 33 ORF576 627912 628607 putative ORF577 628774 629697 putative ORF578 629660 631639 POMP91A U65942 Chlamydia psittaci 579 44 ORF579 631725 633551 putative ORF580 633520 636957 putative 98 kDa outer membrane U72499 Chlamydia psittaci 266 45 protein ORF581 637232 638098 adhesion protein D90903 Synechocystis sp. 267 38 ORF582 640648 639593 GTP-binding protein D90901 Synechocystis sp. 759 45 ORF583 640979 640728 50S ribosomal protein L27 U38804 Porphyra purpurea 265 65 ORF584 641327 641007 50S ribosomal subunit protein L21 U18997 Escherichia coli 210 41 ORF585 641687 642283 hypothetical protein D90906 Synechocystis sp. 76 39 ORF586 643023 642286 assimilatory sulfite reductase L26503 Saccharomyces cerevisiae 284 42 ORF587 643330 643076 putative ORF588 643704 643351 ribosomal protein S10 (rpS10) U32761 Haemophilus influenzae 349 69 ORF589 645628 643676 translation elongation factor EF-G AF000625 Helicobacter pylori 1991 58 (fusA) ORF590 645783 645538 elongation factor G (AA 1-691) X16278 Thermus aquaticus 170 80 thermophilus ORF591 646269 645793 ribosomal protein S7 Z11567 Chlamydia trachomatis 730 88 ORF592 646751 646314 ribosomal protein S12 (AA 1-123) X52912 Cryptomonas phi 485 67 ORF593 647848 647045 putative ORF594 648393 650336 ORF of prc gene (alt.) D00674 Escherichia coli 554 42 ORF595 651016 650420 hypothetical sulfur-rich protein U41759 Chlamydia psittaci 301 50 ORF596 652956 651289 60 kDa CrP X53511 Chlamydia pneumoniae 2951 100 ORF597 653395 653126 9 kDa CrP X53511 Chlamydia pneumoniae 502 99 ORF598 655740 654193 glutamyl-tRNA synthetase homolog U41759 Chlamydia psittaci 2259 82 ORF599 656508 655966 early stage-specific transcription L13598 Chlamydia psittaci 666 62 experimentally demonstrated; early upstream open reading frame (EUO) ORF600 658140 657022 unknown U41759 Chlamydia psittaci 950 44 ORF601 660216 658525 RecJ recombination protein U41759 Chlamydia psittaci 807 73 ORF602 663238 660248 protein-export membrane protein D64000 Synechocystis sp. 413 41 SecD ORF603 664461 663157 putative ORF604 665735 664635 putative ORF605 666212 666994 hypothetical protein D64006 Synechocystis sp. 538 58 ORF606 666998 667921 o298; This 298 aa orf is 33 pct AE000238 Escherichia coli 253 45 identical (24 gaps) to 248 residues of an approx. 256 aa protein CDSA_ECOLI SW: P06466 ORF607 667909 668568 cytidylate kinase AE000193 Escherichia coli 400 48 ORF608 668502 669203 hypothetical protein D90915 Synechocyslis sp. 225 33 ORF609 669154 670893 arginyl-tRNA-synthetase D64006 Synechocystis sp. 1365 49 ORF610 672226 670853 UDP-N-acetylglucosamine U32788 Haemophilus influenzae 642 40 enolpyruvyl transferase (murZ) ORF611 671137 671424 putative ORF612 672453 673001 putative ORF613 673072 674721 putative ORF614 674549 674262 putative ORF615 675518 674796 ORF246 gene product X59551 Escherichia coli 520 43 ORF616 676083 675499 putative ORF617 676630 676067 putative ORF618 677016 676600 ORF3 D10279 Bacillus subtilis 361 63 ORF619 677647 677015 peptide release factor 2 X99401 Bacillus firmus 427 43 ORF620 677990 678259 unknown Z49939 Saccharomyces cerevisiae 175 48 ORF621 679444 680097 unknown D26185 Bacillus subtilis 263 38 ORF622 680097 680897 unknown D64126 Bacillus subtilis 506 45 ORF623 681637 680849 putative ORF624 681409 682281 putative ORF625 682453 682821 putative ORF626 682763 683902 sensor protein L39904 Myxococcus xanthus 190 48 ORF627 684616 683969 putative ORF628 685169 684534 putative ORF629 685986 685117 putative ORF630 686278 687288 NtrC/NifA-like protein regulator U17902 Escherichia coli 820 45 ORF631 687483 688151 putative ORF632 688740 689501 putative ORF633 690242 689622 putative ORF634 690470 691126 unknown Z48008 Saccharomyces cerevisiae 380 46 ORF635 692600 691497 putative ORF636 692674 695064 phenylalanyl-tRNA synthetase U32810 Haemophilus influenzae 593 45 beta-subunit (pheT) ORF637 695049 696032 putative ORF638 697964 696585 OppC-like protein D85103 Synechococcus sp. 371 37 ORF639 699803 698274 OppB gene product X56347 Bacillus subtilis 197 40 ORF640 701926 699788 AppA U20909 Bacillus subtilis 324 43 ORF641 703196 702567 putative ORF642 704221 703208 putative ORF643 704240 705289 ferrochelatase X73417 Arabidopsis thaliana 266 42 ORF644 706070 705300 histidine periplasmic binding protein U58045 Campylobacter jejuni 128 31 P29 ORF645 706841 706254 conserved hypothetical protein AE000592 Helicobacter pylori 155 37 ORF646 707596 706811 putative ORF647 708666 707677 ADP-glucose pyrophosphorylase X55650 Solanum tuberosum 595 43 ORF648 709793 709119 pyrE-F gene product X71842 Arabidopsis thaliana 400 44 ORF649 711523 710132 transcription termination factor J01673 Escherichia coli 1251 60 ORF650 712236 711523 putative ORF651 714734 712125 DNA polymerase I J04479 Streptococcus pneumoniae 1334 43 ORF652 715759 714761 protease IV U67512 Methanococcus jannaschii 101 55 ORF653 717538 715886 adenine nucleotide translocase Z49227 Arabidopsis thaliana 832 39 ORF654 719113 720243 replicative DNA helicase D26185 Bacillus subtilis 776 44 ORF655 720590 722422 homologous to E. coli gidA X62540 Pseudomonas putida 1575 52 ORF656 722406 723056 putative ORF657 723551 723120 nucleoside 5′-diphosphate J05207 Myxococcus xanthus 451 62 phosphotransferase (EC 2.7.4.6) ORF658 724246 723626 Holliday junction DNA helicase U32716 Haemophilus influenzae 293 43 (ruvA) ORF659 724754 724251 crossover junction U32717 Haemophilus influenzae 296 53 endodeoxyribonuclease (ruvC) ORF660 725868 724900 putative ORF661 727115 726270 putative ORF662 728126 727119 glyceraldehyde-3-phosphate U83198 Chlamydia trachomatis 1340 75 dehydrogenase ORF663 728594 728208 ribosomal protein L17 L33834 Chlamydia trachomatis 439 82 ORF664 729614 728604 RNA polymerase alpha-subunit L33834 Chlamydia trachomatis 1356 89 ORF665 729778 729533 RNA polymerase alpha-subunit L33834 Chlamydia trachomatis 273 82 ORF666 730149 729751 ribosomal protein S11 L33834 Chlamydia trachomatis 562 90 ORF667 730539 730174 ribosomal protein S13 L33834 Chlamydia trachomatis 544 89 ORF668 731983 730598 homolog L25077 Chlamydia trachomatis 1956 83 ORF669 732427 731996 ribosomal protein CtrL15e M80325 Chlamydia trachomatis 563 77 ORF670 732917 732423 ribosomal protein CtrS5e M80325 Chlamydia trachomatis 702 84 ORF671 733598 733320 ribosomal protein L6 M60652 Chlamydia trachomatis 316 87 ORF672 733869 733492 ribosomal protein L6 M60652 Chlamydia trachomatis 469 77 ORF673 734298 733900 ribosomal protein CtrS8e M80325 Chlamydia trachomatis 572 82 ORF674 734858 734319 ribosomal protein CtrL5e M80325 Chlamydia trachomatis 730 90 ORF675 735195 734863 ribosomal protein CtrL24e M80325 Chlamydia trachomatis 420 70 ORF676 735578 735342 ribosomal protein CtrL14e M80325 Chlamydia trachomatis 270 95 ORF677 735861 735604 ribosomal protein S17e M80325 Chlamydia trachomatis 322 77 ORF678 736492 736079 50S ribosomal protein L16 D90905 Synechocystis sp. 439 60 ORF679 737192 736524 ribosomal protein S3 D64071 Actinobacillus 612 58 actinomycetemcomitans ORF680 737555 737211 ribosomal protein L22 Z21677 Thermotoga maritima 228 48 ORF681 738688 737837 50S ribosomal subunit protein L2 U18997 Escherichia coli 769 62 ORF682 739048 738713 putative ORF683 739736 739065 ribosomal protein L4 X67014 Bacillus stearothermophilus 308 46 ORF684 740477 739773 ribosomal protein L3 Z46265 Thermus aquaticus 463 50 thermophilus ORF685 740659 740958 putative ORF686 741722 740721 putative ORF687 742789 741827 methionyl-tRNA formyltransferase D64001 Synechocystis sp. 511 48 ORF688 743618 742782 UDP-N-acetylglucosamine L22690 Rickettsia rickettsii 542 43 acyltransferase ORF689 744092 743634 (3R)-hydroxymyristol acyl carrier D90910 Synechocystis sp. 339 55 protein dehydrase ORF690 744604 744107 UDP-3-0-acyl N-acetylglcosamine D90902 Synechocystis sp. 287 45 deacetylase ORF691 744953 744498 UDP-3-O-acyl-GlcNAc deacetylase U67855 Pseudomonas aeruginosa 262 51 ORF692 746608 744986 apolipoprotein N-acyltransferase U32716 Haemophilus influenzae 194 50 (cute) ORF693 747085 746621 low homology to P14 protein of D78189 Bacillus subtilis 235 37 Heamophilus influenzar and 14.2 kDa protein of Escherichia coli ORF694 747974 747219 polymerase III M22996 Bacillus subtilis 180 34 ORF695 748594 748169 hypothetical protein D90914 Synechocystis sp. 160 43 ORF696 749145 748573 putative ORF697 749652 749957 trxA L39892 Chlamydia psittaci 393 72 ORF698 750446 749979 spoU L39892 Chlamydia psittaci 559 72 ORF699 751219 750446 mip L39892 Chlamydia psittaci 948 60 ORF700 753042 751291 aspartyl-tRNA synthetase D90910 Synechocystis sp. 1347 47 ORF701 754309 753020 histidine-tRNA ligase Z17214 Streptococcus equisimilis 757 44 ORF702 755120 756175 hexosephosphate transport protein M89480 Salmonella typhimurium 870 49 ORF703 756120 756485 hexosephosphate transport protein M89479 Escherichia coli 321 45 ORF704 756499 760227 DNA polymerase III alpha-subunit AE000646 Helicobacter pylori 1977 42 (dnaE) ORF705 761217 760297 putative ORF706 761297 761809 putative ORF707 761782 762282 putative ORF708 762260 762895 putative ORF709 762867 763316 hypothetical protein D90908 Synechocystis sp. 177 43 ORF710 763780 763325 putative ORF711 763861 765168 DD-carboxypeptidase M85047 Bacillus subtilis 292 37 ORF712 766809 765697 fmu and fmv protein D90902 Synechocystis sp. 130 36 ORF713 768051 766888 putative ORF714 768566 768321 putative ORF715 769342 768551 putative ORF716 770532 769378 putative ORF717 771451 770804 putative ORF718 773058 771847 3-phosphoglycerate kinase U83197 Chlamydia trachomatis 1540 72 ORF719 773094 773456 putative ORF720 774376 773093 putative phosphate permease U84890 Mesembryanthemum 870 45 crystallinum ORF721 775123 774380 putative ORF722 775398 774916 putative ORF723 775046 776077 sporulation protein M57689 Bacillus subtilis 698 43 ORF724 776070 777041 was dppE U00039 Escherichia coli 565 56 ORF725 777964 777536 orf288; translated orf similarity to Y10436 Coxiella burnetii 256 46 SWISS-PROT: YGI2_PSEPU hypothetical 32.4 kDa protein of Pseudomomas putida ORF726 778176 777904 B. subtilis genes rpmH, rnpA, 50 kd, X62539 Bacillus subtilis 112 37 gidA and gidB ORF727 778621 779334 putative ORF728 781173 780307 f406; This 406 aa orf is 28 pct AE000263 Escherichia coli 603 40 identical (12 gaps) to 264 residues of an approx. 440 aa protein YAOA_SCHPO SW: Q10089 ORF729 781526 781116 f406; This 406 aa orf is 28 pct AE000263 Escherichia coli 258 45 identical (12 gaps) to 264 residues of an approx. 440 aa protein YAOA_SCHPO SW: Q10089 ORF730 782784 781555 f423; This 423 aa orf is 29 pct AE000263 Escherichia coli 197 44 identical (1 gaps) to 172 residues of an approx. 488 aa protein YC24_CYAPA SW: P48260 ORF731 783572 782805 hypothetical chloroplast ORF16 U38804 Porphyra purpurea 597 52 ORF732 785032 783581 ABC transporter subunit D64004 Synechocystis sp. 1720 62 ORF733 786412 785360 putative ORF734 788429 786450 pbp Y14206 Streptomyces coelicolor 148 55 ORF735 788944 788528 penicillin-binding protein 3 X84053 Pseudomonas aeruginosa 148 38 ORF736 789758 788901 putative ORF737 790332 791504 major outer membrane protein M64064 Chlamydia pneumoniae 2028 99 ORF738 791846 792721 ribosomal protein S2 U60196 Chlamydia trachomatis 904 70 ORF739 792724 793569 elongation factor Ts U60196 Chlamydia trachomatis 1023 71 ORF740 793580 794323 UMP kinase U60196 Chlamydia trachomatis 891 72 ORF741 794304 794843 ribosome-releasing factor U60196 Chlamydia trachomatis 673 73 ORF742 795217 795732 unknown D26185 Bacillus subtilis 105 42 ORF743 795722 796795 unknown D26185 Bacillus subtilis 208 33 ORF744 798735 797053 putative L33796 Vibrio cholerae 386 34 ORF745 799823 798681 putative ORF746 799297 799578 putative ORF747 801313 799808 Pkn5 U40656 Myxococcus xanthus 345 33 ORF748 802453 801332 putative ORF749 803299 802457 putative ORF750 803811 803290 putative ORF751 805151 803826 YscN U02499 Yersinia enterocolitica 1185 53 ORF752 805860 805156 putative ORF753 806604 806332 putative ORF754 806913 806608 putative ORF755 808222 806903 putative ORF756 808751 808146 putative ORF757 809437 808673 putative ORF758 809939 809454 putative ORF759 811235 810213 delta-aminolevulinate synthase (EC M30785 Escherichia coli 172 40 2.3.1.37) ORF760 811779 813056 DNA gyrase subunit B U35453 Clostridium acetobutylicum 584 38 ORF761 812890 812516 putative ORF762 812954 813583 DNA gyrase subunit B Z19108 Spiroplasma citri 371 39 ORF763 813587 815023 gyrA X92503 Mycobacterium smegmatis 414 55 ORF764 815420 815746 putative ORF765 816036 817010 orf-X; hypothetical protein; Method: U48870 Bacillus subtilis 569 47 conceptual translation supplied by author ORF766 817111 817356 unknown Z74024 Mycobacterium tuberculosis 114 34 ORF767 817791 818609 3-deoxy-d-manno-octulosonic acid Z50747 Chlamydia psittaci 1112 78 8-phosphate synthetase ORF768 818609 819094 protein of unknown function Z50747 Chlamydia psittaci 545 65 ORF769 819104 819823 ATP binding protein U72493 Chlamydia trachomatis 1099 88 ORF770 820722 819826 putative ORF771 822313 821000 putative ORF772 823503 822238 putative ORF773 823678 825612 putative ORF774 825461 826312 putative ORF775 827280 826645 putative ORF776 828604 827171 76 kDa protein L23921 Chlamydia pneumoniae 2179 100 ORF777 830026 828713 76 kDa protein L23921 Chlamydia pneumoniae 1162 100 ORF778 831047 830085 mviB homolog U50732 Chlamydia trachomatis 982 58 ORF779 831725 831051 mviB homolog U50732 Chlamydia trachomatis 740 65 ORF780 832220 833098 T05H10.2 Z47812 Caenorhabditis elegans 407 34 ORF781 833851 833396 ribosomal protein S4 (rps4) AE000633 Helicobacter pylori 372 53 ORF782 834068 835039 This ORF is homologous to a 40.0 kd L22217 Mycoplasma-like organism 377 49 hypothetical protein in the htrB 3′ region from E. coli, Accession Number X61000 ORF783 835792 835127 uridine kinase L31783 Mus musculus 436 43 ORF784 837624 836116 ORF_f397 U29581 Escherichia coli 92 38 ORF785 838951 840882 putative ORF786 840869 842185 exodeoxyribonuclease V (recB) U32811 Haemophilus influenzae 409 40 ORF787 841989 843455 DNA helicase II U39703 Mycoplasma genitalium 110 46 ORF788 843242 844021 exodeoxyribonuclease V (recB) U32811 Haemophilus influenzae 196 40 ORF789 845018 843987 MreC protein M31792 Escherichia coli 76 53 ORF790 846174 844990 aspartate aminotransferase (aspC) X03629 Escherichia coli 754 40 ORF791 848509 846311 GreA U02878 Rickettsia prowazekii 190 35 ORF792 848568 849014 putative ORF793 849082 850488 NADH: ubiquinone oxidoreducatase U32702 Haemophilus influenzae 445 37 subunit A (GP: Z37111_2) ORF794 851512 850574 porphobilinogen synthase U38348 Chlorobium vibrioforme 769 45 ORF795 852064 852447 putative ORF796 852398 853690 putative ORF797 855118 854243 geranylgeranyl pyrophosphate D85029 Arabidopsis thaliana 408 41 synthase ORF798 855751 855128 f147; This 147 aa orf is 26 pct AE000143 Escherichia coli 187 36 identical (1 gaps) to 99 residues of an approx. 728 aa protein E2BE_RABIT SW: P47823 ORF799 856551 855829 membrane associated regulatory M28368 Salmonella typhimurium 172 36 protein ORF800 856730 858556 unknown function Z32530 Chlamydia trachomatis 842 35 ORF801 858717 859601 exodeoxyribonuclease V (recD) U32811 Haemophilus influenzae 182 51 ORF802 859591 860205 exonuclease V alpha subunit (AA X04582 Escherichia coli 235 45 1-608) ORF803 861132 860284 putative ORF804 861426 861163 30S ribosomal protein S20 Z67753 Odontella sinensis 153 41 ORF805 861701 862921 putative ORF806 863026 864798 major sigma factor U04442 Chlamydia psiitaci 2661 94 ORF807 864831 865256 putative ORF808 865226 866581 dihydropterin pyrophosphokinase/ Y08611 Pisum sativum 455 48 dihydropteroate synthase ORF809 866562 867119 dehydrofolate reductase, type I U32772 Haemophilus influenzae 213 49 (folA) ORF810 867025 867816 M. jannaschii predicted coding U67522 Methanococcus jannaschii 207 36 region MJ0768 ORF811 867820 868497 putative ORF812 869743 868661 RecA U16739 Chlamydia trachomatis 1512 87 ORF813 870633 870094 unknown function Z32530 Chlamydia trachomatis 308 45 ORF814 871929 870646 unknown function Z32530 Chlamydia trachomatis 1410 63 ORF815 872538 872086 putative ORF816 873908 872517 putative ORF817 874281 874670 nifR3-like gene product Z37984 Azospirillum brasilense 181 32 ORF818 874582 875286 ORF1 gene product X62399 Escherichia coli 307 42 ORF819 877857 875377 DNA topoisomerase I L27797 Bacillis subtilis 1488 50 ORF820 878446 879255 putative ORF821 880635 879268 sigma factor (ntrA) (AA 1-502) X05888 Azotobacter vinelandii 257 47 ORF822 882524 880593 DNA helicase II D90906 Synechocystis sp. 1140 50 ORF823 882612 883319 ipa-57d gene product X73124 Bacillus subtilis 601 51 ORF824 884155 883538 hypothetical protein D90915 Synechocystis sp. 344 39 ORF825 884340 885611 19/20 residue stretch (32-51) L19954 Bacillus subtilis 456 37 identical to N-terminal putative signal sequence of unknown, partly cloned B. subtilis gene.; putative ORF826 885722 887302 heat shock protein L12004 Chlamydia trachomatis 915 39 ORF827 887587 888153 bas1 protein Z34917 Hordeum vulgare 474 50 ORF828 888627 888220 putative ORF829 889330 888716 hypothetical protein Y14079 Bacillus subtilis 223 55 ORF830 889898 889323 peptidoglycan-associated lipoprotein X65796 Escherichia coli 222 50 ORF831 891190 889898 TolB U32470 Haemophilus influenzae 280 35 ORF832 891828 891247 putative ORF833 892421 892017 exbD peptide M28819 Escherichia coli 77 48 ORF834 893116 892421 inner membrane protein (tolQ) U32722 Haemophilus influenzae 157 54 ORF835 892521 892925 putative ORF836 893392 895419 inner membrane copper tolerance Z36905 Escherichia coli 120 35 protein ORF837 895745 896527 unknown D26185 Bacillus subtilis 381 41 ORF838 896668 897558 succinate dehydrogenase subunit C Y08563 Paenibacillus macerans 253 40 ORF839 897565 899442 succinate dehydrogenase subunit A Y08563 Paenibacillus macerans 1667 57 ORF840 899420 900229 succinate dehydrogenase subunit B Y08563 Paenibacillus macerans 656 54 ORF841 903230 900237 putative ORF842 905081 903234 putative ORF843 906931 905045 sigma factor SibG regulation protein D90905 Synechocystis sp. 117 35 RsbU ORF844 907248 907832 putative ORF845 907784 908128 putative ORF846 908132 908677 putative ORF847 908589 909320 putative ORF848 909405 911465 putative ORF849 911677 912360 putative ORF850 912303 912821 putative ORF851 912937 913983 putative ORF852 915128 914067 putative ORF853 916658 915303 enolase L29475 Bacillus subtilis 1036 60 ORF854 915627 915376 enolase U43738 Mycoplasma pneumoniae 226 65 ORF855 917707 916853 excinuclease ABC subunit B (uvrB) U32804 Haemophilus influenzae 724 46 ORF856 918837 917722 excinuclease ABC subunit B (uvrB) U32804 Haemophilus influenzae 1029 54 ORF857 919868 918837 tryptophanyl-tRNA synthetase (trpS) U32746 Haemophilus influenzae 376 40 ORF858 920434 919880 putative ORF859 921187 920438 ORF8 X82078 Chlamydia sp. 164 50 ORF860 921959 921195 hypothetical protein X62475 Chlamydia psittaci 511 44 ORF861 923773 921995 Threonyl tRNA Synthetase Z80360 Bacillus subtilis 1476 44 ORF862 922146 922415 putative ORF863 923943 923674 putative ORF864 924077 925006 putative ORF865 925436 925083 putative ORF866 926524 925349 putative ORF867 927920 926433 putative ORF868 928319 927951 putative ORF869 928963 928334 putative ORF870 929248 930987 DNA mismatch repair protein U32692 Haemophilus influenzae 585 40 (mutL) ORF871 930995 932059 YqhT D84432 Bacillus subtilis 445 39 ORF872 932121 933515 putative ORF873 932881 932513 putative ORF874 933485 935746 pulD (ttg start codon) M32613 Klebsiella pneumoniae 210 33 ORF875 935724 937082 epsE M96172 Vibrio cholerae 890 55 ORF876 937229 938410 PilG U32588 Neisseria gonorrhoeae 280 38 ORF877 938281 938805 putative ORF878 938809 939255 putative ORF879 939165 939782 putative ORF880 939760 940791 putative ORF881 940822 941106 putative ORF882 940977 941351 putative ORF883 942537 941623 yscT L25667 Yersinia pseudotuberculosis 169 44 ORF884 942784 942500 yscS L25667 Yersinia pseudotuberculosis 173 42 ORF885 943149 942799 HrcR AE000107 Rhizobium sp. NGR234 265 52 ORF886 943799 943029 pathogenicity protein M64094 Xanthomonas campestris 252 41 ORF887 944055 943732 putative M74011 Yersinia enterocolitica 112 33 ORF888 944413 943994 putative ORF889 945395 944556 putative ORF890 945853 945389 putative ORF891 946392 945751 HrcJ U56662 Erwinia amylovora 229 44 ORF892 947410 948081 putative ORF893 949871 948915 ORF YOR196c Z75104 Saccharomyces cerevisiae 702 44 ORF894 951058 949868 dihydrolipoamide dehydrogenase E3 M57435 Bacillus subtilis 745 39 subunit ORF895 951249 950959 dihydrolipoamide acetyltransferase M73535 Staphylococcus aureus 166 49 E3 subunit ORF896 951664 952134 putative ORF897 952674 952165 SNF X98455 Bacillus aereus 229 47 ORF898 953491 952589 helicase U39680 Mycoplasma genitalium 307 42 ORF899 955324 953495 F01G4.1 Z68341 Caenorhabditis elegans 133 57 ORF900 955823 955281 putative ORF901 957082 955847 branched-chain amino acid carrier Z48676 Lactobacillus delbrueckii 297 40 ORF902 957902 957270 endonuclease III U11289 Bacillus subtilis 317 37 ORF903 959231 957906 homologous to E. coli 50K X62539 Bacillus subtilis 805 45 ORF904 959376 960284 phosphatidylserine decarboxylase U72715 Chlamydia trachomatis 776 51 ORF905 960266 961669 putative ORF906 961856 964765 secretory component U06928 Caulobacter crescentus 1812 55 ORF907 966855 965395 28.2% of identity to the Escherichia L47648 Bacillus subtilis 778 41 coli GTP-binding protein Era; putative ORF908 968204 966975 poly(A) polymerase L47709 Bacillus subtilis 383 41 ORF909 968791 968237 ClpX-like protein U18229 Bacillus subtilis 340 39 ORF910 969498 968731 ATP-dependent protease ATPase D64006 Synechocystis sp. 846 66 subunit ORF911 969858 969511 ClpP U16135 Synechococcus sp. 257 54 ORF912 970118 969762 ATP-dependent clp protease AE000591 Helicobacter pylori 362 63 proteolytic component (clpP) ORF913 970593 970300 putative ORF914 971261 970542 putative ORF915 971680 971123 putative ORF916 971876 975100 SNF X98455 Bacillus cereus 778 49 ORF917 975419 976516 MreB protein M96343 Bacillus subtilis 960 55 ORF918 976584 978320 phospho enol pyruvate S56812 Chlorobium limicola 1667 64 carboxykinase ORF919 977680 977231 putative ORF920 978399 980738 putative ORF921 980756 981928 putative ORF922 982974 981931 precursor protein (AA-22 to 371) X52557 Chlamydia trachomatis 97 50 ORF923 984120 983119 NAD+ dependent L47648 Bacillus subtilis 618 43 glycerol-3-phosphate dehydrogenase ORF924 985502 984120 AgX-1 antigen [human, infertile S73498 Homo sapiens 254 34 patient, testis, Peptide, 505 aa] ORF925 987180 985882 ORF4 M72718 Bacillus subtilis 697 38 ORF926 987172 987444 putative ORF927 989846 989049 nifU-like protein AE000542 Helicobacter pylori 302 31 ORF928 991048 989846 putative ORF929 991638 990955 phosphoglyceromutase L09651 Zymomonas mobilis 471 53 ORF930 991794 992498 ORFX13 L09228 Bacillus subtilis 403 39 ORF931 993619 993041 biotin [acetyl-COA-carboxylase] L47709 Bacillus subtilis 136 38 ligase ORF932 993530 994792 rod-shape-determining protein M22857 Escherichia coli 312 44 ORF933 995970 994795 cadmium-transporting ATPase D64005 Synechocystis sp. 358 47 ORF934 996857 995739 ATPase L28104 Transposon Tn5422 449 39 ORF935 997603 996782 putative ORF936 998969 997572 seryl-trna synthetase Y09924 Staphylococcus aureus 851 42 ORF937 998896 1000023 orf2, homologue to B. subtilis ribG X64395 Escherichia coli 596 40 ORF938 1000087 1001340 GTP cyclohydrolase II D90912 Synechocystis sp. 1078 52 ORF939 1001357 1001818 riboflavin synthase beta subunit U27202 Actinobacillus 278 36 pleuropneumoniae ORF940 1003288 1001873 putative ORF941 1003487 1004146 putative ORF942 1004485 1005639 D-alanine glycine permease (dagA) AE000603 Helicobacter pylori 394 33 ORF943 1005643 1005972 hypothetical protein MTCY180.08 Z97193 Mycobacterium tuberculosis 274 58 ORF944 1006784 1006116 similar to trithorax protein in final U13875 Caenorhabditis elegans 155 46 three exons ORF945 1007563 1006769 yycJ D78193 Bacillus subtilis 406 38 ORF946 1009226 1007568 YtpT AF008220 Bacillus subtilis 992 47 ORF947 1009989 1009336 putative ORF948 1015852 1016337 putative ORF949 1016561 1016181 putative ORF950 1016297 1017532 putative ORF951 1016802 1016452 putative ORF952 1018993 1017701 phenolhydroxylase component U32702 Haemophilus influenzae 909 47 ORF953 1019454 1019137 ORF M63939 Escherichia coli 96 45 ORF954 1020764 1019562 pCTHom1 gene product M94254 Chlamydia trachomatis 1185 65 ORF955 1021405 1021037 histone H1-like protein M80324 Chlamydia psittaci 319 62 ORF956 1021821 1024286 phosphoprotein L25078 Chlamydia trachomatis 739 41 ORF957 1024697 1024248 putative ORF958 1025569 1024508 protoporphyrinogen oxidase U25114 Mus musculus 86 38 ORF959 1026969 1025590 oxygen independent D90912 Synechocystis sp. 880 42 coprophorphyrinogen III oxidase ORF960 1027789 1026947 uroporphyrinogen decarboxylase M97208 Bacillus subtilis 372 38 ORF961 1031199 1027945 transcription-repair coupling factor U32805 Haemophilus influenzae 1584 42 (trcF) (mfd) ORF962 1031717 1031172 alanyl-tRNA synthetase X95571 Thiobacillus ferrooxidans 76 31 ORF963 1033057 1031612 alanyl-tRNA synthetase AE000353 Escherichia coli 889 40 ORF964 1033425 1033039 alanyl-tRNA synthetase (alaS) AE000629 Helicobacter pylori 327 51 ORF965 1033784 1033200 alanyl-tRNA synthetase X59956 Rhizobium leguminosarum 416 47 ORF966 1033963 1036038 transketolase Z73234 Bacillus subtilis 1398 44 ORF967 1036945 1036010 AMP nucleosidase AE000290 Escherichia coli 265 42 ORF968 1037110 1037679 elongation factor P U140003 Escherichia coli 458 51 ORF969 1037696 1037944 putative ORF970 1038916 1037975 putative ORF971 1040582 1039026 HSP60 chaperonin X62914 Clostridium perfringens 284 31 ORF972 1040997 1042337 PROBABLE AB001488 Bacillus subtilis 446 39 UDP-N-ACETYLMURAMOYLAL ANYL-D-GLUTAMYL-2, 6-DIAMINOLIGASE (EC 6.3.2.15). ORF973 1042357 1043403 ORF-Y (AA 1-360) X51584 Escherichia coli 582 45 ORF974 1043367 1044623 UDP-N-acetylmuramoylalanine-D-gl U32793 Haemophilus influenzae 348 42 utamate ligase (murD) ORF975 1044607 1045362 hypothetical protein Y14079 Bacillus subtilis 115 38 ORF976 1045384 1046538 spoVE gene product (AA 1-366) X51419 Bacillus subtilis 479 35 ORF977 1046447 1047517 mur Y13922 Enterococcus hirae 256 45 ORF978 1047521 1049956 UDP-N-acetylmuramate-alanine U32794 Haemophilus influenzae 756 38 ligase (murC) ORF979 1050611 1050036 unknown Z74024 Mycobacterium tuberculosis 78 44 ORF980 1050925 1050566 cycY gene product U14003 Escherichia coli 179 34 ORF981 1051728 1051090 putative ORF982 1051743 1052063 hypothetical protein D90908 Synechocystis sp. 135 33 ORF983 1052101 1053126 trna Z98209 Mycobacterium tuberculosis 441 37 delta(2)-isopentenylpyrophosphate transferase ORF984 1054201 1053107 conserved hypothetical protein AE000579 Helicobacter pylori 826 44 ORF985 1054242 1055555 putative ORF986 1055483 1055908 putative ORF987 1056609 1056965 YqeL D84432 Bacillus subtilis 202 38 ORF988 1056961 1058232 beta-ketoacyl-ACP synthase L13242 Ricinus communis 1266 55 ORF989 1058238 1058687 diadenosine tetraphosphatase U30313 Homo sapiens 122 42 ORF990 1059371 1058727 inorganic pyrophosphatase (ppa) AE000576 Helicobacter pylori 209 39 ORF991 1059526 1060578 leucine dehydrogenase LeuDH U51099 Bacillus cereus 680 45 ORF992 1061553 1060579 3′(2′),5′-bisphosphate nucleotidase U40433 Arabidopsis thaliana 335 43 ORF993 1061674 1062411 putative ORF994 1062377 1064077 2-acylglycerophosphoethanolamine U29581 Escherichia coli 383 44 acyl transferase/acyl carrier protein synthetase ORF995 1064116 1065243 7-keto-8-aminopelargonic acid M29291 Bacillus sphaericus 200 35 synthetase (bioF) ORF996 1067451 1065178 priA Y10304 Bacillus subtilis 1009 43 ORF997 1068065 1067376 putative ORF998 1068209 1068706 putative ORF999 1069958 1068819 unknown U41759 Chlamydia psittaci 777 41 ORF1000 1071163 1070033 unknown U41759 Chlamydia psittaci 381 36 ORF1001 1072438 1071332 unknown U41759 Chlamydia psittaci 254 37 ORF1002 1072997 1073476 putative ORF1003 1074239 1075864 lysyl-tRNA synthetase D90906 Synechocystis sp. 1007 48 ORF1004 1076790 1075867 cysteinyl-tRNA synthetase L14580 Bacillus subtilis 395 52 ORF1005 1077268 1076573 cys-tRNA synthetase (cysS) U32693 Haemophilus influenzae 431 56 ORF1006 1077999 1078724 putative ORF1007 1079088 1078672 ribonuclease P protein component M11056 Escherichia coli 78 46 (gtg start codon) ORF1008 1079642 1079944 30S ribosomal subunit protein S14 U18997 Escherichia coli 260 50 ORF1009 1080501 1079995 F18C12.2 Z75536 Caenorhabditis elegans 118 38 ORF1010 1080775 1081341 putative ORF1011 1083158 1081350 deoxyribodipyrimidine photolyase J03294 Bacillus subtilis 687 44 ORF1012 1084677 1083235 DNA mismatch repair protein U71154 Aquifex pyrophilus 735 48 ORF1013 1085648 1084632 DNA mismatch repair protein D90909 Synechocystis sp. 565 39 ORF1014 1086117 1086737 DNA primase (dnaG) U32735 Haemophilus influenzae 303 40 ORF1015 1086692 1087897 DnaG Z83860 Mycobacterium tuberculosis 222 37 ORF1016 1088646 1089005 putative ORF1017 1089146 1089805 putative ORF1018 1092931 1089890 glycyl-tRNA synthetase U20547 Chlamydia trachomatis 2569 48 ORF1019 1093179 1092889 putative ORF1020 1093584 1094204 phosphatidylglycerophosphate U87792 Bacillus subtilis 163 55 synthase ORF1021 1095619 1094192 glycogen (starch) synthase D90899 Synechocystis sp. 574 40 ORF1022 1096074 1096628 partial ctc gene product (AA 1-186) X16518 Bacillus subtilis 86 37 ORF1023 1096633 1097082 peptidyl-tRNA hydrolase U31570 Chlamydia trachomatis 378 53 ORF1024 1097266 1097601 ribosomal protein S6 (rps6) AE000630 Helicobacter pylori 179 39 ORF1025 1097622 1097867 ribosomal protein S18 homolog; M62820 Chlamydia trachomatis 324 86 putative ORF1026 1097886 1098392 putative heat shock protein ORF; M62820 Chlamydia trachomatis 190 79 putative ORF1027 1699521 1099279 putative ORF1028 1099689 1101053 putative ORF1029 1102192 1101107 putative ORF1030 1104950 1102116 glycerol-3-phosphate acyltransferase M80571 Cucumis sativus 574 43 ORF1031 1106508 1104946 ORF_f495; orfF of ECMRED, uses U18997 Escherichia coli 855 38 2nd start ORF1032 1106722 1107249 putative ORF1033 1107463 1108101 PlsX U59433 Bacillus subtilis 282 45 ORF1034 1108041 1108421 fatty acid/phospholipid synthesis AE000540 Helicobacter pylori 205 35 protein (plsX) ORF1035 1108520 1113370 putative 98 kDa outer membrane U72499 Chlamydia psittaci 352 44 protein ORF1036 1114958 1113447 putative ORF1037 1116915 1115071 lipid A disaccharide synthetase U32786 Haemophilus influenzae 477 42 (lpxB) ORF1038 1118183 1116894 poly(A) polymerase AE000123 Escherichia coli 555 46 ORF1039 1118846 1120030 putative L12968 Escherichia coli 880 50 ORF1040 1120040 1120522 glucosamine fructose-6-phosphate AE000651 Helicobacter pylori 396 52 aminotransferase (isomerizing) (glmS) ORF1041 1120510 1121430 glutamine amidotransferase; AE000450 Escherichia coli 494 44 glucosamine-fructose-6-phosphate aminotransferase ORF1042 1121321 1121866 L-glutamine: D-fructose-6-P U17352 Thermus aquaticus 374 50 amidotransferase precursor thermophilus ORF1043 1122123 1122899 tyrosine-specific transport protein AE000284 Escherichia coli 281 41 ORF1044 1124842 1125564 putative ORF1045 1126526 1125579 cell division protein (ftsY) U32760 Haemophilus influenzae 497 41 ORF1046 1126519 1127676 succinyl-CoA synthetase J01619 Escherichia coli 784 43 beta-subunit ORF1047 1127672 1128571 succinyl coenzyme A synthetase U23408 Dictyostelium discoideum 978 63 alpha subunit ORF1048 1130230 1131336 putative ORF1049 1131480 1132553 putative ORF1050 1132830 1133843 putative ORF1051 1134121 1134855 serine protease HtrA D90905 Synechocystis sp. 307 51 ORF1052 1134642 1135592 GsrA protein D78376 Yersinia enterocolitica 497 41 ORF1053 1135964 1135653 putative ORF1054 1137132 1135954 R11H6.1 Z93386 Caenorhabditis elegans 445 37 ORF1055 1137169 1140102 Ydr430cp; CAI: 0.15 U33007 Saccharomyces cerevisiae 559 40 ORF1056 1141365 1140112 hypothetical 54.7 kD protein in udp AE000459 Escherichia coli 222 34 3′ region precursor (o475) ORF1057 1142150 1141356 phosphatidylserine synthase (pssA) AE000614 Helicobacter pylori 307 41 ORF1058 1142520 1145660 ribonucleotide reductase subunit M1 K02927 Mus musculus 1433 45 ORF1059 1145627 1146721 ribonucleoside diphosphate AE000553 Helicobacter pylori 443 32 reductase, beta subunit (nrdB) ORF1060 1146862 1147545 unknown Z95398 Mycobacterium leprae 191 35 ORF1061 1147666 1148190 YtqB AF008220 Bacillus subtilis 262 44 ORF1062 1148514 1148224 ORF2 U01958 Bacillus licheniformis 135 54 ORF1063 1149136 1148348 ORF2 M31827 Bacillus subtilis 268 40 ORF1064 1149702 1149166 putative ORF1065 1150031 1150591 unknown Z85982 Mycobacterium tuberculosis 445 49 ORF1066 1150785 1151147 ribosomal protein L20 (AA 1-119) X16188 Bacillus stearothermophilus 273 44 ORF1067 1151165 1152181 phenylalany-tRNA synthetase beta Z75208 Bacillus subtilis 777 40 subunit ORF1068 1152522 1154591 putative ORF1069 1155666 1154566 putative ORF1070 1156743 1155670 putative ORF1071 1156859 1157815 hypothetical U32723 Haemophilus influenzae 252 42 ORF1072 1157982 1160735 ATP-binding protein U01376 Escherichia coli 1314 56 ORF1073 1162620 1160917 polynucleotide phosphorylase AF010578 Pisum sativum 1416 52 ORF1074 1162970 1162590 polyribonucleotide phophorylase U52048 Spinacia oleracea 312 53 ORF1075 1163532 1164020 orf150 gene product X95938 Porphyromonas gingivalis 335 43 ORF1076 1163995 1164294 putative ORF1077 1165569 1165030 putative ORF1078 1166108 1165566 putative ORF1079 1166644 1166141 putative ORF1080 1167055 1168374 putative ORF1081 1169218 1168337 methionine aminopeptidase D64003 Synechocystis sp. 488 54 ORF1082 1169823 1169218 ORF_o197 U18997 Escherichia coli 281 30 ORF1083 1171324 1170572 putative ORF1084 1172085 1171177 hypothetical U32720 Haemophilus influenzae 162 44 ORF1085 1172394 1173773 fumarase D64000 Synechocystis sp. 1292 57 ORF1086 1175209 1173881 prs-associated putative membrane U02424 Escherichia coli 570 39 protein ORF1087 1175555 1175127 hypothetical protein in pth-prs AE000219 Escherichia coli 278 46 intergenic region ORF1088 1175778 1177043 hypothetical protein Z96072 Mycobacterium tuberculosis 109 43 ORF1089 1177177 1179048 putative ORF1090 1179156 1180085 penicillin tolerance protein (lytB) U32781 Haemophilus influenzae 731 54 ORF1091 1180045 1180779 putative ORF1092 1181942 1180788 putative ORF1093 1182296 1181961 putative ORF1094 1183844 1182300 putative ORF1095 1184420 1183848 putative ORF1096 1185382 1184366 putative ORF1097 1185858 1185226 putative ORF1098 1186164 1186481 putative ORF1099 1187386 1186484 site-specific recombinase U92524 Salmonella typhimurium 401 48 ORF1100 1187370 1189028 phophoglucoisomerase-like protein L40822 Chlamydia trachomatis 1154 63 ORF1101 1189321 1190889 putative ORF1102 1191142 1192146 NADP-malate dehydrogenase L40958 Flaveria bidentis 775 46 ORF1103 1191974 1191729 putative ORF1104 1193815 1192991 putative ORF1105 1195702 1194248 o460; This 460 aa orf is 46 pct AE000256 Escherichia coli 1022 44 identical (26 gaps) to 458 residues of an approx. 488 aa protein ARCD_PSEAE SW: P18275 ORF1106 1196303 1195716 putative ORF1107 1196831 1196337 putative ORF1108 1197807 1196746 putative ORF1109 1198740 1197883 putative ORF1110 1200232 1198721 shikimate 5-dehydrogenase U67551 Methanococcus jannaschii 245 37 ORF1111 1201286 1200135 3-dehydroquinate synthase (aroB) U32705 Haemophilus influenzae 478 45 ORF1112 1202386 1201259 2,3-dihydroxybenzoic acid L29562 Vibrio anguillarum 780 50 ORF1113 1202901 1202350 putative ORF1114 1204162 1202816 5-enolpyruvylshikimate 3-phosphate U67500 Methanococcus jannaschii 520 40 synthase ORF1115 1203177 1203464 putative ORF1116 1205028 1204180 putative ORF1117 1206392 1204878 bioA gene product A02587 unidentified 834 48 ORF1118 1206742 1206086 dethiobiotin synthase (bioD) U32830 Haemophilus influenzae 243 37 ORF1119 1207872 1206724 L-alanine-pimelyl CoA ligase U51868 Bacillus subtilis 601 41 ORF1120 1208852 1207851 biotin sythase U24147 Arabidopsis thaliana 892 52 ORF1121 1210518 1209742 tryptophan hydroxylase U26428 Gallus gallus 237 34 ORF1122 1210703 1211494 dihydrodipicolinate reductase U47017 Pseudomonas syringae pv. 345 37 tabaci ORF1123 1211870 1212754 aspartate-semialdehyde U67476 Methanococcus jannaschii 444 43 dehydrogenase ORF1124 1212742 1214064 aspartokinase III U00006 Escherichia coli 473 47 ORF1125 1214046 1214858 dihydrodipicolinate synthase D64006 Synechocystis sp. 238 40 ORF1126 1215551 1216318 putative ORF1127 1216493 1216849 putative ORF1128 1217183 1219612 putative ORF1129 1220068 1219673 putative ORF1130 1219710 1220669 putative ORF1131 1220630 1221376 putative ORF1132 1221645 1223681 unknown D26185 Bacillus subtilis 621 43 ORF1133 1223894 1224988 putative ORF1134 1225000 1225830 high level kasgamycin resistance D26185 Bacillus subtilis 422 41 ORF1135 1227810 1225879 hypothetical protein D90903 Synechocystis sp. 1129 43 ORF1136 1226528 1226908 putative ORF1137 1229972 1228311 exonuclease VII, large subunit U32723 Haemophilus influenzae 666 46 (xseA) ORF1138 47569 47018 Integrase/recombinase AE001308 Chlamydia trachomatis 716 72 ORF1139 49980 49117 putative ORF1140 53356 52898 putative ORF1141 54477 54884 O-Sialoglycoprotein Endopeptidase AE001307 Chlamydia trachomatis 311 51 ORF1142 63753 63998 PTS PEP Phosphotransferase AE001306 Chlamydia trachomatis 198 61 ORF1143 77164 77487 putative ORF1144 79724 79302 Sms Protein AE001302 Chlamydia trachomatis 458 57 ORF1145 88721 88951 putative ORF1146 94067 94429 putative ORF1147 122832 123341 hypothetical protein AE001303 Chlamydia trachomatis 398 61 ORF1148 147536 147234 putative ORF1149 158990 159346 S16 Ribosomal Protein AE001277 Chlamydia trachomatis 467 78 ORF1150 168470 168979 putative ORF1151 169183 169452 putative ORF1152 171785 171504 Cationic Amino Acid Transporter AE001278 Chlamydia trachomatis 262 68 ORF1153 172518 171775 Cationic Amino Acid Transporter AE001278 Chlamydia trachomatis 533 48 ORF1154 193599 194045 putative ORF1155 195704 196075 S/T Protein Kinase AE001288 Chlamydia trachomatis 536 82 ORF1156 210687 210145 KDO-transferase X80061 Chlamydia pneumoniae 856 96 ORF1157 211100 210708 putative ORF1158 215420 215088 putative ORF1159 217914 218246 putative ORF1160 218925 218701 putative ORF1161 223785 223525 IMP dehydrogenase U13372 Borrelia burgdorferi 270 63 ORF1162 224271 223999 putative ORF1163 228691 228407 putative ORF1164 235050 235334 (Methylase) AE001287 Chlamydia trachomatis 331 66 ORF1165 252308 253021 Oligopeptide Permease AE001293 Chlamydia trachomatis 838 72 ORF1166 258280 258912 Dicarboxylate Translocator AE001294 Chlamydia trachomatis 909 80 ORF1167 261325 261567 putative ORF1168 268195 268878 hypothetical protein AE001287 Chlamydia trachomatis 556 52 ORF1169 269447 268881 putative ORF1170 271263 271538 putative ORF1171 271957 272346 putative ORF1172 274176 274550 putative ORF1173 275736 275314 Disulfide bond Oxidoreductase AE001291 Chlamydia trachomatis 519 73 ORF1174 276490 276927 hypothetical protein AE001291 Chlamydia trachomatis 249 53 ORF1175 277577 277861 hypothetical protein AE001291 Chlamydia trachomatis 256 52 ORF1176 288163 287909 putative ORF1177 290130 289789 putative ORF1178 290989 291225 putative ORF1179 291372 291860 adenylate cyclase AE001286 Chlamydia trachomatis 388 48 ORF1180 311239 311622 putative ORF1181 328665 328384 putative ORF1182 337348 338289 sodium-dependent transporter AF017105 Chlamydia psittaci 1112 72 ORF1183 364764 364369 Prolipoprotein Diacylglycerol AE001298 Chlamydia trachomatis 300 54 Transferase ORF1184 389623 390135 hypothetical protein AE001282 Chlamydia trachomatis 75 33 ORF1185 393729 394343 ABC superfamily ATPase AE001282 Chlamydia trachomatis 473 52 ORF1186 407379 407621 putative ORF1187 410944 410708 putative ORF1188 427632 427988 putative ORF1189 428172 428486 putative ORF1190 436761 437246 hypothetical protein AE001279 Chlamydia trachomatis 661 81 ORF1191 460911 461159 putative ORF1192 477597 477313 hypothetical protein AE001300 Chlamydia trachomatis 309 62 ORF1193 487303 487001 putative ORF1194 487764 487534 Glycine Cleavage System H Protein AE001300 Chlamydia trachomatis 221 67 ORF1195 498502 499017 hypothetical protein AE001275 Chlamydia trachomatis 206 32 ORF1196 499795 500466 putative ORF1197 571928 572344 putative ORF1198 572367 572131 putative ORF1199 588184 587915 hypothetical protein AE001312 Chlamydia trachomatis 256 62 ORF1200 600587 600907 (Metalloenzyme) AE001316 Chlamydia trachomatis 314 61 ORF1201 609731 608895 putative ORF1202 614039 614755 hypothetical protein AE001317 Chlamydia trachomatis 475 46 ORF1203 614823 615152 putative ORF1204 638244 638831 ABC Transporter ATPase AE001315 Chlamydia trachomatis 614 61 ORF1205 638819 639094 (Metal Transport Protein) AE001315 Chlamydia trachomatis 265 63 ORF1206 639073 639636 (Metal Transport Protein) AE001315 Chlamydia trachomatis 687 69 ORF1207 647901 648236 hypothetical protein AE001317 Chlamydia trachomatis 139 38 ORF1208 678510 679469 phosphohydrolase AE001320 Chlamydia trachomatis 995 63 ORF1209 688178 688732 hypothetical protein AE001320 Chlamydia trachomatis 366 43 ORF1210 696045 696563 methyltransferase AE001321 Chlamydia trachomatis 369 49 ORF1211 708998 708588 Glucose-1-P Adenyltransferase AE001322 Chlamydia trachomatis 507 83 ORF1212 709808 710089 putative ORF1213 718240 717737 Glycerol-3-P Phosphatidyltransferase AE001323 Chlamydia trachomatis 573 66 ORF1214 737828 737565 S19 Ribosomal Protein AE001323 Chlamydia trachomatis 439 94 ORF1215 779502 780257 hypothetical protein AE001322 Chlamydia trachomatis 476 48 ORF1216 806310 805864 hypothetical protein AE001337 Chlamydia trachomatis 512 67 ORF1217 820931 820707 putative ORF1218 837696 839096 Exodeoxyribonuclease V, Gamma AE001334 Chlamydia trachomatis 967 49 ORF1219 883307 883549 putative ORF1220 892010 891726 putative ORF1221 893277 893564 putative ORF1222 936998 937225 Gen. Secretion Protein E AE001327 Chlamydia trachomatis 256 67 ORF1223 946865 947419 putative ORF1224 975187 975411 SWF/SNF family helicase AE001341 Chlamydia trachomatis 363 96 ORF1225 985882 985517 hypothetical protein AE001342 Chlamydia trachomatis 166 33 ORF1226 987713 987180 hypothetical protein AE001342 Chlamydia trachomatis 447 59 ORF1227 988215 987733 Flagellar M-Ring Protein AE001342 Chlamydia trachomatis 304 44 ORF1228 988754 988530 Flagellar M-Ring Protein AE001342 Chlamydia trachomatis 92 36 ORF1229 992542 992841 hypothetical protein AE001343 Chlamydia trachomatis 112 39 ORF1230 992759 993067 hypothetical protein AE001343 Chlamydia trachomatis 100 32 ORF1231 1004247 1004528 D-Ala/Gly Permease AE001344 Chlamydia trachomalis 283 64 ORF1232 1015013 1014294 235aa long hypothetical protein AB009472 Pyrococcus horikoshii 104 54 ORF1233 1056147 1056545 putative ORF1234 1077682 1078035 predicted disulfide bond isomerase AE001351 Chlamydia trachomatis 233 46 ORF1235 1088121 1088381 putative ORF1236 1098430 1098852 Predicted Kinase AE001352 Chlamydia trachomatis 384 59 ORF1237 1098798 1099319 Predicted Kinase AE001352 Chlamydia trachomatis 322 45 ORF1238 1123198 1123515 Transport Permease AE001354 Chlamydia trachomatis 313 72 ORF1239 1123606 1124256 Tyrosine Transport AE001354 Chlamydia trachomatis 577 58 ORF1240 1124453 1124797 Tyrosine Transport AE001354 Chlamydia trachomalis 323 50 ORF1241 1129253 1129567 putative ORF1242 1164947 1164474 hypothetical protein AE001357 Chlamydia trachomatis 412 56 ORF1243 1170457 1170053 hypothetical protein AE001358 Chlamydia trachomatis 283 59 ORF1244 1172342 1171863 ABC transporter permease AE001358 Chlamydia trachomatis 457 55 ORF1245 1192155 1192835 putative ORF1246 1192759 1192992 putative ORF1247 1193861 1194142 putative ORF1248 1194036 1193779 (D-Amino Acid Dehydrogenase) AE001311 Chlamydia trachomatis 269 79 ORF1249 1209748 1209053 conserved hypothetical protein AE000958 Archaeoglobus fulgidus 121 38 ORF1250 1215111 1215419 putative ORF1251 1216302 1216538 putative ORF1252 1228072 1227818 hypothetical protein AE001306 Chlamydia trachomatis 134 39 ORF1253 1228304 1228080 xseB AL021897 Mycobacterium tuberculosis 89 33 ORF1254 26599 26222 putative ORF1255 27609 27367 putative ORF1256 67206 66967 putative ORF1257 70612 70352 putative ORF1258 132703 132945 putative ORF1259 178073 178393 putative ORF1260 208576 208349 putative ORF1261 209156 208929 putative ORF1262 209263 209024 putative ORF1263 210304 210639 putative ORF1264 299009 299452 putative ORF1265 352106 351717 putative ORF1266 420182 419949 Flagellar Secretion Protein AE001280 Chlamydia trachomatis 115 43 ORF1267 553602 553381 putative ORF1268 556538 556807 putative ORF1269 594348 593797 putative ORF1270 595169 594876 putative ORF1271 662148 662381 putative ORF1272 706528 706893 putative ORF1273 803315 803650 putative ORF1274 849551 849306 putative ORF1275 913676 913275 putative ORF1276 927087 926836 putative ORF1277 930587 930360 putative ORF1278 986531 986764 ORF12 M72718 Bacillus subtilis 106 48 ORF1279 996229 996486 putative ORF1280 1000373 1000002 putative ORF1281 1010291 1010037 putative ORF1282 1011128 1010793 106 aa long hypothetical protein AB009472 Pyrococcus horikoshii 159 50 ORF1283 1012924 1012694 putative ORF1284 1028659 1028913 putative ORF1285 1086481 1086762 putative ORF1286 1118658 1118879 Phosphoglucomutase AE001354 Chlamydia trachomatis 291 84 ORF1287 1170098 1169835 hypothetical protein AE001358 Chlamydia trachomatis 187 53 ORF1288 1180828 1181184 putative ORF1289 1182658 1183035 putative ORF1290 1195076 1194795 putative ORF1291 1195890 1196183 putative

TABLE 2 ORF Nos begin end potential start 2 42 794 42 3 1258 1614 1261 4 1807 2418 1807 5 3393 2491 3393 6 3639 4067 3639 7 5649 4270 5649 8 7463 6012 7463 9 8051 8962 8051 10 9129 9959 9138 11 10687 10361 10639 12 10927 11232 10927 13 11246 12727 11246 14 12691 14190 12691 15 14484 17249 14484 16 16039 15770 16036 17 17845 20853 17845 18 21137 22042 21137 19 22046 23476 22046 20 23681 26110 23681 21 26109 25861 26109 22 26241 26978 26241 23 26960 27754 26960 24 27747 28577 27747 25 28887 29492 28950 26 29432 30028 29432 27 30024 31472 30024 28 31758 32288 31758 29 32201 33991 32201 30 33852 34541 33852 31 34783 36063 34783 32 36009 37529 36009 33 37881 39362 37881 34 39418 39161 39418 35 39366 40715 39366 36 43076 41094 43076 37 43800 43066 43800 38 44828 43785 44768 39 45340 44753 45340 40 45752 45372 45752 41 46996 45701 46996 42 47961 47569 47961 43 48960 48040 48960 44 51452 50133 51452 45 52606 51335 52606 46 53684 53319 53684 47 54195 53746 54195 48 55278 56453 55278 49 56493 57266 56493 50 57297 58526 57297 51 59851 58565 59851 52 61495 59924 61495 53 61324 62151 61324 54 62132 62470 62132 55 62474 63733 62474 56 63881 64186 63881 57 64611 64318 64611 58 65485 64673 65485 59 65999 65301 65999 60 66244 67281 66244 61 67265 67699 67265 62 67703 68539 67760 63 68805 70736 68805 64 69172 68831 69172 65 70642 71142 70642 66 71325 72029 71325 67 72060 73637 72060 68 74061 76175 74061 69 78351 77680 78351 70 79356 78355 79356 71 79983 79693 79983 72 80441 79938 80441 73 80475 80969 80475 74 81296 83080 81332 75 83291 83932 83291 76 84005 84769 84005 77 84975 85244 84975 78 85123 85425 85123 79 85397 85903 85397 80 85909 86583 85909 81 86626 88065 86626 82 89257 91026 89257 83 91291 93030 91291 84 93295 94086 93295 85 95285 94707 95279 86 95667 96557 95667 87 96317 97456 96317 88 98435 97968 98435 89 99460 98426 99460 90 100144 101325 100144 91 101457 101720 101457 92 101704 102273 101704 93 102356 102805 i02356 94 102835 103530 102835 95 103549 104058 103549 96 104096 104491 104096 97 104601 108386 104601 98 108401 112054 108401 99 112033 112590 112033 100 112672 113682 112672 101 113726 114121 113726 102 114711 114136 114711 103 115267 115755 115267 104 115911 116543 115911 105 116736 118055 116778 106 117968 118522 117968 107 118530 119843 118530 108 119816 120457 119816 109 120451 122430 120451 110 122504 122950 122504 111 123528 126347 123528 112 126332 129166 126332 113 134690 129213 134690 114 134925 136382 134931 115 137870 136482 137867 116 137899 138240 137899 117 138239 137928 138239 118 139558 138257 139558 119 140352 139516 140352 120 140498 141841 140498 121 141855 142658 141855 122 144258 143050 144258 123 145258 144494 145258 124 145454 146749 145454 125 147318 146767 147318 126 148261 147677 148261 127 149029 152157 149029 128 154108 152201 154108 129 155135 154308 155135 130 155141 155467 155141 131 155703 156779 155703 132 156748 157635 156748 133 157653 158996 157653 134 159363 159986 159363 135 159880 160446 159880 136 160477 160839 160477 137 160898 161539 160898 138 161527 162153 161527 139 162144 162443 162144 140 162437 164098 162437 141 165451 164228 165451 142 166349 165411 166349 143 166949 168442 166949 144 169416 171029 169416 145 170857 171459 170857 146 172652 173428 172652 147 174626 173439 174626 148 174816 175613 174816 149 175598 175954 175598 150 175958 176935 175958 151 177708 176938 177708 152 177128 177376 177128 153 179472 177841 179472 154 179822 179517 179822 155 181793 179943 181793 156 182628 181876 182628 157 184420 183074 184420 158 184988 184467 184988 159 185483 185112 185483 160 185902 185483 185902 161 186174 185839 186174 162 187720 186587 187720 163 188318 190933 188318 164 191090 191635 191090 165 191547 192743 191547 166 192969 193469 192969 167 194044 193610 194044 168 194196 195809 194196 169 196088 198073 196088 170 198132 199454 198132 171 199351 202818 199351 172 204552 202999 204552 173 205648 204692 205639 174 205807 207327 205807 175 207182 207775 207182 176 207779 208267 207779 177 208267 209577 208267 178 211807 211271 211807 179 212188 211844 212188 180 214079 212448 214079 181 214907 214083 214907 182 216154 215429 216154 183 216115 216678 216115 184 216728 217282 216728 185 217267 217866 217267 186 218593 218261 218590 187 219821 218994 219821 188 221382 220309 221382 189 222719 221433 222719 190 223521 222724 223521 191 224499 225008 224499 192 225140 225559 225140 193 225555 226802 225555 194 227800 226892 227743 195 228335 228072 228335 196 229251 228643 229251 197 230983 229622 230983 198 231483 230983 231483 199 232063 231509 232063 200 232739 232053 232739 201 233166 234356 233166 202 233518 233165 233518 203 234536 235186 234536 204 235379 236689 235379 205 236680 237618 236689 206 237521 238345 237521 207 238281 238973 238281 208 238871 240115 238871 209 240191 241564 240191 210 242281 241604 242281 211 242933 242274 242933 212 243416 242976 243416 213 243500 244531 243500 214 244480 246021 244480 215 246330 247811 246330 216 247831 249174 247870 217 249437 251038 249455 218 251325 252212 251325 219 253156 254007 253156 220 253974 254852 253974 221 255258 256094 255258 222 256640 257455 256640 223 257502 258239 257502 224 257869 257501 257869 225 259248 260897 259248 226 262753 261788 262753 227 263059 262757 263059 228 264375 263182 264375 229 265985 264747 265985 230 266637 266059 266637 231 267338 266538 267338 232 267922 267473 267922 233 269647 270771 269647 234 272777 273145 272777 235 273253 273636 273253 236 273705 273977 273705 237 276016 275717 276016 238 276439 276020 276418 239 276792 277253 276792 240 277318 277599 277318 241 278578 277877 278578 242 279258 278554 279258 243 280435 279533 280435 244 281547 280849 281547 245 281696 282325 281717 246 282459 284069 282459 247 284056 284517 284056 248 284606 285775 284606 249 285592 285987 285592 250 286179 286976 286179 251 287583 287002 287583 252 287951 287451 287951 253 288499 288816 288499 254 289674 288505 289674 255 288839 289213 288839 256 289970 290254 289970 257 291931 292803 291931 258 293258 292755 293258 259 293718 293272 293718 260 294630 293953 294630 261 296153 294636 296153 262 294817 295068 294817 263 296354 297862 296354 264 298415 297879 298415 265 298777 298253 298777 266 299572 298781 299572 267 300487 299633 300487 268 301586 300702 301568 269 302440 301571 302440 270 302838 302437 302838 271 303335 302745 303335 272 304394 303852 304394 273 304606 305223 304606 274 305394 306236 305394 275 306501 307439 306501 276 308033 307458 308033 277 308924 308037 308924 278 309485 310180 309485 279 310426 311214 310426 280 311597 311253 311504 281 312772 311780 312772 282 313425 312772 313425 283 313646 313377 313646 284 313937 314665 313937 285 315576 314755 315576 286 316157 315531 316157 287 318657 316156 318657 288 321042 318676 321042 289 321445 321098 321445 290 322309 321710 322309 291 323190 322366 323181 292 323843 323181 323843 293 324878 323856 324878 294 325340 326410 325340 295 326433 327836 326433 296 328465 327839 328465 297 329360 328857 329360 298 330907 329357 330907 299 332455 330956 332455 300 334536 332395 334536 301 336091 334877 336091 302 336103 337302 336103 303 338129 338830 338129 304 338965 339501 338965 305 339508 340143 339508 306 340247 342967 340247 307 343385 343810 343385 308 344171 343935 344171 309 345082 344330 345073 310 346005 345082 346005 311 346784 346437 346784 312 347029 346715 347029 313 347034 347723 347034 314 348075 350459 348075 315 350598 351071 350598 316 351075 352175 351096 317 353291 352230 353267 318 353442 354467 353442 319 354451 354933 354451 320 355000 355449 355000 321 355448 356743 355448 322 355953 355642 355953 323 359310 356827 359310 324 359120 359377 359120 325 359525 359908 359525 326 361290 359947 361290 327 363785 361362 363746 328 364496 363888 364496 329 364832 365290 364832 330 365304 365669 365304 331 366599 365667 366599 332 367291 369030 367291 333 369134 369808 369134 334 369917 370438 369917 335 370365 372647 370365 336 372557 373066 372557 337 373020 373442 373020 338 373467 374195 373467 339 374176 375099 374176 340 375676 375083 375676 341 376173 375634 376173 342 376564 377643 376564 343 377956 379773 377956 344 379781 380425 379805 345 380281 381000 380281 346 381008 381460 381008 347 381460 383037 381460 348 383257 383523 383257 349 383553 385304 383553 350 385397 386458 385400 351 387242 386514 387242 352 388764 387013 388764 353 390120 390932 390120 354 390919 391818 390961 355 392379 391885 392379 356 392582 392986 392582 357 392776 393684 392776 358 394151 394804 394151 359 394928 395308 394928 360 395259 395990 395259 361 397815 395953 397815 362 398850 397831 398850 363 400085 399099 400085 364 401245 400073 401236 365 401474 401136 401474 366 402199 401423 402199 367 403193 402186 403166 368 403650 404165 403650 369 404343 405914 404343 370 405984 407327 405984 371 407712 408806 407712 372 410439 409075 410439 373 411826 410954 411826 374 412482 414302 412482 375 415402 414407 415402 376 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1078672 1079088 1008 1079642 1079944 1079642 1009 1080501 1079995 1080468 1010 1080775 1081341 1080775 1011 1083158 1081350 1083158 1012 1084677 1083235 1084677 1013 1085648 1084632 1085648 1014 1086117 1086737 1086117 1015 1086692 1087897 1086692 1016 1088646 1089005 1088646 1017 1089146 1089805 1089146 1018 1092931 1089890 1092931 1019 1093179 1092889 1093179 1020 1093584 1094204 1093584 1021 1095619 1094192 1095619 1022 1096074 1096628 1096074 1023 1096633 1097082 1096633 1024 1097266 1097601 1097266 1025 1097622 1097867 1097622 1026 1097886 1098392 1097886 1027 1099521 1099279 1099521 1028 1099689 1101053 1099704 1029 1102192 1101107 1102192 1030 1104950 1102116 1104950 1031 1106508 1104946 1106508 1032 1106722 1107249 1106722 1033 1107463 1108101 1107463 1034 1108041 1108421 1108041 1035 1108520 1113370 1108520 1036 1114958 1113447 1114958 1037 1116915 1115071 1116915 1038 1118183 1116894 1118183 1039 1118846 1120030 1118846 1040 1120040 1120522 1120040 1041 1120510 1121430 1120510 1042 1121321 1121866 1121321 1043 1122123 1122899 1122123 1044 1124842 1125564 1124842 1045 1126526 1125579 1126526 1046 1126519 1127676 1126519 1047 1127672 1128571 1127672 1048 1130230 1131336 1130230 1049 1131480 1132553 1131480 1050 1132830 1133843 1132830 1051 1134121 1134855 1134121 1052 1134642 1135592 1134642 1053 1135964 1135653 1135964 1054 1137132 1135954 1137132 1055 1137169 1140102 1137169 1056 1141365 1140112 1141344 1057 1142150 1141356 1142150 1058 1142520 1145660 1142520 1059 1145627 1146721 1145627 1060 1146862 1147545 1146862 1061 1147666 1148190 1147666 1062 1148514 1148224 1148514 1063 1149136 1148348 1149136 1064 1149702 1149166 1149702 1065 1150031 1150591 1150031 1066 1150785 1151147 1150785 1067 1151165 1152181 1151165 1068 1152522 1154591 1152522 1069 1155666 1154566 1155666 1070 1156743 1155670 1156740 1071 1156859 1157815 1156859 1072 1157982 1160735 1157982 1073 1162620 1160917 1162620 1074 1162970 1162590 1162970 1075 1163532 1164020 1163532 1076 1163995 1164294 1163995 1077 1165569 1165030 1165569 1078 1166108 1165566 1166108 1079 1166644 1166141 1166644 1080 1167055 1168374 1167055 1081 1169218 1168337 1169218 1082 1169823 1169218 1169823 1083 1171324 1170572 1171324 1084 1172085 1171177 1172085 1085 1172394 1173773 1172394 1086 1175209 1173881 1175209 1087 1175555 1175127 1175360 1088 1175778 1177043 1175778 1089 1177177 1179048 1177177 1090 1179156 1180085 1179156 1091 1180045 1180779 1180045 1092 1181942 1180788 1181942 1093 1182296 1181961 1182296 1094 1183844 1182300 1183844 1095 1184420 1183848 1184420 1096 1185382 1184366 1185382 1097 1185858 1185226 1185858 1098 1186164 1186481 1186185 1099 1187386 1186484 1187386 1100 1187370 1189028 1187370 1101 1189321 1190889 1189321 1102 1191142 1192146 1191142 1103 1191974 1191729 1191974 1104 1193815 1192991 1193815 1105 1195702 1194248 1195702 1106 1196303 1195716 1196303 1107 1196831 1196337 1196831 1108 1197807 1196746 1197651 1109 1198740 1197883 1198668 1110 1200232 1198721 1200232 1111 1201286 1200135 1201286 1112 1202386 1201259 1202350 1113 1202901 1202350 1202901 1114 1204162 1202816 1204162 1115 1203177 1203464 1203177 1116 1205028 1204180 1205028 1117 1206392 1204878 1206392 1118 1206742 1206086 1206742 1119 1207872 1206724 1207872 1120 1208852 1207851 1208852 1121 1210518 1209742 1210518 1122 1210703 1211494 1210703 1123 1211870 1212754 1211870 1124 1212742 1214064 1212742 1125 1214046 1214858 1214046 1126 1215551 1216318 1215551 1127 1216493 1216849 1216493 1128 1217183 1219612 1217183 1129 1220068 1219673 1220068 1130 1219710 1220669 1219710 1131 1220630 1221376 1220630 1132 1221645 1223681 1221645 1133 1223894 1224988 1223900 1134 1225000 1225830 1225000 1135 1227810 1225879 1227810 1136 1226528 1226908 1226528 1137 1229972 1228311 1229972 1138 47569 47018 47569 1139 49980 49117 49980 1140 53356 52898 53356 1141 54477 54884 54477 1142 63753 63998 63753 1143 77164 77487 77164 1144 79724 79302 79724 1145 88721 88951 88721 1146 94067 94429 94067 1147 122832 123341 122832 1148 147536 147234 147536 1149 158990 159346 158990 1150 168470 168979 168470 1151 169183 169452 169204 1152 171785 171504 171785 1153 172518 171775 172518 1154 193599 194045 193599 1155 195704 196075 195704 1156 210687 210145 210684 1157 211100 210708 211100 1158 215420 215088 215420 1159 217914 218246 217914 1160 218925 218701 218925 1161 223785 223525 223785 1162 224271 223999 224271 1163 228691 228407 228691 1164 235050 235334 235050 1165 252308 253021 252308 1166 258280 258912 258280 1167 261325 261567 261325 1168 268195 268878 268195 1169 269447 268881 269447 1170 271263 271538 271263 1171 271957 272346 271957 1172 274176 274550 274176 1173 275736 275314 275736 1174 276490 276927 276490 1175 277577 277861 277577 1176 288163 287909 288163 1177 290130 289789 290130 1178 290989 291225 290989 1179 291372 291860 291372 1180 311239 311622 311239 1181 328665 328384 328665 1182 337348 338289 337348 1183 364764 364369 364764 1184 389623 390135 389623 1185 393729 394343 393729 1186 407379 407621 407379 1187 410944 410708 410944 1188 427632 427988 427632 1189 428172 428486 428172 1190 436761 437246 436761 1191 460911 461159 460911 1192 477597 477313 477597 1193 487303 487001 487303 1194 487764 487534 487764 1195 498502 499017 498502 1196 499795 500466 499795 1197 571928 572344 571928 1198 572367 572131 572367 1199 588184 587915 588184 1200 600587 600907 600587 1201 609731 608895 609731 1202 614039 614755 614039 1203 614823 615152 614823 1204 638244 638831 638244 1205 638819 639094 638819 1206 639073 639636 639073 1207 647901 648236 647901 1208 678510 679469 678510 1209 688178 688732 688178 1210 696045 696563 696045 1211 708998 708588 708998 1212 709808 710089 709808 1213 718240 717737 718240 1214 737828 737565 737828 1215 779502 780257 779502 1216 806310 805864 806310 1217 820931 820707 820931 1218 837696 839096 837696 1219 883307 883549 883307 1220 892010 891726 892010 1221 893277 893564 893277 1222 936998 937225 936998 1223 946865 947419 946865 1225 985882 985517 985882 1224 975187 975411 975187 1226 987713 987180 987713 1227 988215 987733 988215 1228 988754 988530 988754 1229 992542 992841 992542 1230 992759 993067 992759 1231 1004247 1004528 1004268 1232 1015013 1014294 1015013 1233 1056147 1056545 1056147 1234 1077682 1078035 1077682 1235 1088121 1088381 1088121 1236 1098430 1098852 1098430 1237 1098798 1099319 1098798 1238 1123198 1123515 1123198 1239 1123606 1124256 1123606 1240 1124453 1124797 1124453 1241 1129253 1129567 1129253 1242 1164947 1164474 1164947 1243 1170457 1170053 1170457 1244 1172342 1171863 1172342 1245 1192155 1192835 1192155 1246 1192759 1192992 1192759 1247 1193861 1194142 1193861 1248 1194036 1193779 1194036 1249 1209748 1209053 1209748 1250 1215111 1215419 1215111 1251 1216302 1216538 1216302 1252 1228072 1227818 1228072 1253 1228304 1228080 1228304 1254 26599 26222 26599 1255 27609 27367 27609 1256 67206 66967 67197 1257 70612 70352 70588 1258 132703 132945 132703 1259 178073 178393 178073 1260 208576 208349 208576 1261 209156 208929 209156 1262 209263 209024 209263 1263 210304 210639 210304 1264 299009 299452 299030 1265 352106 351717 352061 1266 420182 419949 420170 1267 553602 553381 553602 1268 556538 556807 556538 1269 594348 593797 594342 1270 595169 594876 595160 1271 662148 662381 662160 1272 706528 706893 706528 1273 803315 803650 803339 1274 849551 849306 849551 1275 913676 913275 913676 1276 927087 926836 927087 1277 930587 930360 930587 1278 986531 986764 986531 1279 996229 996486 996229 1280 1000373 1000002 1000334 1281 1010291 1010037 1010273 1282 1011128 1010793 1011128 1283 1012924 1012694 1012924 1284 1028659 1028913 1028659 1285 1086481 1086762 1086481 1286 1118658 1118879 1118658 1287 1170098 1169835 1170098 1288 1180828 1181184 1180828 1289 1182658 1183035 1182658 1290 1195076 1194795 1195055 1291 1195890 1196183 1195890 1292 189042 188809 189030 1293 691250 691567 691250 1294 914544 914780 914556 1295 928525 928833 928579 1296 1040685 1040948 1040712 1297 377646 378068 377646

TABLE 4 SEQ ID NO (ORF) Fp Fd Bp Bd 2 1292 1293 3796 3797 3 1294 1295 3798 3799 4 1296 1297 3800 3801 5 1298 1299 3802 3803 6 1300 1301 3804 3805 7 1302 1303 3806 3807 8 1304 1305 3808 3809 9 1306 1307 3810 3811 10 1308 1309 3812 3813 11 1310 1311 3814 3815 12 1312 1313 3816 3817 13 1314 1315 3818 3819 14 1316 1317 3820 3821 15 1318 1319 3822 3823 16 1320 1321 3824 3825 17 1322 1323 3826 3827 18 1324 1325 3828 3829 19 1326 1327 3830 3831 20 1328 1329 3832 3833 21 1330 1331 3834 3835 22 1332 1333 3836 3837 23 1334 1335 3838 3839 24 1336 1337 3840 3841 25 1338 1339 3842 3843 26 1340 1341 3844 3845 27 1342 1343 3846 3847 28 1344 1345 3848 3849 29 1346 1347 3850 3851 30 1348 1349 3852 3853 31 1350 1351 3854 3855 32 1352 1353 3856 3857 33 1354 1355 3858 3859 34 1358 1359 3862 3863 35 1356 1357 3860 3861 36 1360 1361 3864 3865 37 1362 1363 3866 3867 38 1364 1365 3868 3869 39 1366 1367 3870 3871 40 1368 1369 3872 3873 41 1370 1371 3874 3875 42 1374 1375 3878 3879 43 1376 1377 3880 3881 44 1380 1381 3884 3885 45 1382 1383 3886 3887 46 1386 1387 3890 3891 47 1388 1389 3892 3893 48 1392 1393 3896 3897 49 1394 1395 3898 3899 50 1396 1397 3900 3901 51 1398 1399 3902 3903 52 1402 1403 3906 3907 53 1400 1401 3904 3905 54 1404 1405 3908 3909 55 1406 1407 3910 3911 56 1410 1411 3914 3915 57 1412 1413 3916 3917 58 1414 1415 3918 3919 59 1416 1417 3920 3921 60 1418 1419 3922 3923 61 1420 1421 3924 3925 62 1422 1423 3926 3927 63 1424 1425 3928 3929 64 1426 1427 3930 3931 65 1428 1429 3932 3933 66 1430 1431 3934 3935 67 1432 1433 3936 3937 68 1434 1435 3938 3939 69 1438 1439 3942 3943 70 1440 1441 3944 3945 71 1444 1445 3948 3949 72 1446 1447 3950 3951 73 1448 1449 3952 3953 74 1450 1451 3954 3955 75 1452 1453 3956 3957 76 1454 1455 3958 3959 77 1456 1457 3960 3961 78 1458 1459 3962 3963 79 1460 1461 3964 3965 80 1462 1463 3966 3967 81 1464 1465 3968 3969 82 1468 1469 3972 3973 83 1470 1471 3974 3975 84 1472 1473 3976 3977 85 1476 1477 3980 3981 86 1478 1479 3982 3983 87 1480 1481 3984 3985 88 1482 1483 3986 3987 89 1484 1485 3988 3989 90 1486 1487 3990 3991 91 1488 1489 3992 3993 92 1490 1491 3994 3995 93 1492 1493 3996 3997 94 1494 1495 3998 3999 95 1496 1497 4000 4001 96 1498 1499 4002 4003 97 1500 1501 4004 4005 98 1502 1503 4006 4007 99 1504 1505 4008 4009 100 1506 1507 4010 4011 101 1508 1509 4012 4013 102 1510 1511 4014 4015 103 1512 1513 4016 4017 104 1514 1515 4018 4019 105 1516 1517 4020 4021 106 1518 1519 4022 4023 107 1520 1521 4024 4025 108 1522 1523 4026 4027 109 1524 1525 4028 4029 110 1526 1527 4030 4031 111 1530 1531 4034 4035 112 1532 1533 4036 4037 113 1534 1535 4038 4039 114 1536 1537 4040 4041 115 1538 1539 4042 4043 116 1540 1541 4044 4045 117 1542 1543 4046 4047 118 1544 1545 4048 4049 119 1546 1547 4050 4051 120 1548 1549 4052 4053 121 1550 1551 4054 4055 122 1552 1553 4056 4057 123 1554 1555 4058 4059 124 1556 1557 4060 4061 125 1558 1559 4062 4063 126 1562 1563 4066 4067 127 1564 1565 4068 4069 128 1566 1567 4070 4071 129 1568 1569 4072 4073 130 1570 1571 4074 4075 131 1572 1573 4076 4077 132 1574 1575 4078 4079 133 1576 1577 4080 4081 134 1580 1581 4084 4085 135 1582 1583 4086 4087 136 1584 1585 4088 4089 137 1586 1587 4090 4091 138 1588 1589 4092 4093 139 1590 1591 4094 4095 140 1592 1593 4096 4097 141 1594 1595 4098 4099 142 1596 1597 4100 4101 143 1598 1599 4102 4103 144 1604 1605 4108 4109 145 1606 1607 4110 4111 146 1612 1613 4116 4117 147 1614 1615 4118 4119 148 1616 1617 4120 4121 149 1618 1619 4122 4123 150 1620 1621 4124 4125 151 1624 1625 4128 4129 152 1622 1623 4126 4127 153 1626 1627 4130 4131 154 1628 1629 4132 4133 155 1630 1631 4134 4135 156 1632 1633 4136 4137 157 1634 1635 4138 4139 158 1636 1637 4140 4141 159 1638 1639 4142 4143 160 1640 1641 4144 4145 161 1642 1643 4146 4147 162 1644 1645 4148 4149 163 1646 1647 4150 4151 164 1648 1649 4152 4153 165 1650 1651 4154 4155 166 1652 1653 4156 4157 167 1656 1657 4160 4161 168 1658 1659 4162 4163 169 1662 1663 4166 4167 170 1664 1665 4168 4169 171 1666 1667 4170 4171 172 1668 1669 4172 4173 173 1670 1671 4174 4175 174 1672 1673 4176 4177 175 1674 1675 4178 4179 176 1676 1677 4180 4181 177 1678 1679 4182 4183 178 1684 1685 4188 4189 179 1686 1687 4190 4191 180 1688 1689 4192 4193 181 1690 1691 4194 4195 182 1694 1695 4198 4199 183 1696 1697 4200 4201 184 1698 1699 4202 4203 185 1700 1701 4204 4205 186 1704 1705 4208 4209 187 1708 1709 4212 4213 188 1710 1711 4214 4215 189 1712 1713 4216 4217 190 1714 1715 4218 4219 191 1720 1721 4224 4225 192 1722 1723 4226 4227 193 1724 1725 4228 4229 194 1726 1727 4230 4231 195 1728 1729 4232 4233 196 1732 1733 4236 4237 197 1734 1735 4238 4239 198 1736 1737 4240 4241 199 1738 1739 4242 4243 200 1740 1741 4244 4245 201 1742 1743 4246 4247 202 1744 1745 4248 4249 203 1746 1747 4250 4251 204 1750 1751 4254 4255 205 1752 1753 4256 4257 206 1754 1755 4258 4259 207 1756 1757 4260 4261 208 1758 1759 4262 4263 209 1760 1761 4264 4265 210 1762 1763 4266 4267 211 1764 1765 4268 4269 212 1766 1767 4270 4271 213 1768 1769 4272 4273 214 1770 1771 4274 4275 215 1772 1773 4276 4277 216 1774 1775 4278 4279 217 1776 1777 4280 4281 218 1778 1779 4282 4283 219 1782 1783 4286 4287 220 1784 1785 4288 4289 221 1786 1787 4290 4291 222 1788 1789 4292 4293 223 1790 1791 4294 4295 224 1792 1793 4296 4297 225 1796 1797 4300 4301 226 1800 1801 4304 4305 227 1802 1803 4306 4307 228 1804 1805 4308 4309 229 1806 1807 4310 4311 230 1808 1809 4312 4313 231 1810 1811 4314 4315 232 1812 1813 4316 4317 233 1818 1819 4322 4323 234 1824 1825 4328 4329 235 1826 1827 4330 4331 236 1828 1829 4332 4333 237 1834 1835 4338 4339 238 1836 1837 4340 4341 239 1840 1841 4344 4345 240 1842 1843 4346 4347 241 1846 1847 4350 4351 242 1848 1849 4352 4353 243 1850 1851 4354 4355 244 1852 1853 4356 4357 245 1854 1855 4358 4359 246 1856 1857 4360 4361 247 1858 1859 4362 4363 248 1860 1861 4364 4365 249 1862 1863 4366 4367 250 1864 1865 4368 4369 251 1866 1867 4370 4371 252 1868 1869 4372 4373 253 1872 1873 4376 4377 254 1876 1877 4380 4381 255 1874 1875 4378 4379 256 1878 1879 4382 4383 257 1886 1887 4390 4391 258 1888 1889 4392 4393 259 1890 1891 4394 4395 260 1892 1893 4396 4397 261 1896 1897 4400 4401 262 1894 1895 4398 4399 263 1898 1899 4402 4403 264 1900 1901 4404 4405 265 1902 1903 4406 4407 266 1904 1905 4408 4409 267 1906 1907 4410 4411 268 1908 1909 4412 4413 269 1910 1911 4414 4415 270 1912 1913 4416 4417 271 1914 1915 4418 4419 272 1916 1917 4420 4421 273 1918 1919 4422 4423 274 1920 1921 4424 4425 275 1922 1923 4426 4427 276 1924 1925 4428 4429 277 1926 1927 4430 4431 278 1928 1929 4432 4433 279 1930 1931 4434 4435 280 1934 1935 4438 4439 281 1936 1937 4440 4441 282 1938 1939 4442 4443 283 1940 1941 4444 4445 284 1942 1943 4446 4447 285 1944 1945 4448 4449 286 1946 1947 4450 4451 287 1948 1949 4452 4453 288 1950 1951 4454 4455 289 1952 1953 4456 4457 290 1954 1955 4458 4459 291 1956 1957 4460 4461 292 1958 1959 4462 4463 293 1960 1961 4464 4465 294 1962 1963 4466 4467 295 1964 1965 4468 4469 296 1966 1967 4470 4471 297 1970 1971 4474 4475 298 1972 1973 4476 4477 299 1974 1975 4478 4479 300 1976 1977 4480 4481 301 1978 1979 4482 4483 302 1980 1981 4484 4485 303 1984 1985 4488 4489 304 1986 1987 4490 4491 305 1988 1989 4492 4493 306 1990 1991 4494 4495 307 1992 1993 4496 4497 308 1994 1995 4498 4499 309 1996 1997 4500 4501 310 1998 1999 4502 4503 311 2000 2001 4504 4505 312 2002 2003 4506 4507 313 2004 2005 4508 4509 314 2006 2007 4510 4511 315 2008 2009 4512 4513 316 2010 2011 4514 4515 317 2012 2013 4516 4517 318 2014 2015 4518 4519 319 2016 2017 4520 4521 320 2018 2019 4522 4523 321 2020 2021 4524 4525 322 2022 2023 4526 4527 323 2026 2027 4530 4531 324 2024 2025 4528 4529 325 2028 2029 4532 4533 326 2030 2031 4534 4535 327 2032 2033 4536 4537 328 2034 2035 4538 4539 329 2038 2039 4542 4543 330 2040 2041 4544 4545 331 2042 2043 4546 4547 332 2044 2045 4548 4549 333 2046 2047 4550 4551 334 2048 2049 4552 4553 335 2050 2051 4554 4555 336 2052 2053 4556 4557 337 2054 2055 4558 4559 338 2056 2057 4560 4561 339 2058 2059 4562 4563 340 2060 2061 4564 4565 341 2062 2063 4566 4567 342 2064 2065 4568 4569 343 2066 2067 4570 4571 344 2068 2069 4572 4573 345 2070 2071 4574 4575 346 2072 2073 4576 4577 347 2074 2075 4578 4579 348 2076 2077 4580 4581 349 2078 2079 4582 4583 350 2080 2081 4584 4585 351 2082 2083 4586 4587 352 2084 2085 4588 4589 353 2088 2089 4592 4593 354 2090 2091 4594 4595 355 2092 2093 4596 4597 356 2094 2095 4598 4599 357 2096 2097 4600 4601 358 2100 2101 4604 4605 359 2102 2103 4606 4607 360 2104 2105 4608 4609 361 2106 2107 4610 4611 362 2108 2109 4612 4613 363 2110 2111 4614 4615 364 2112 2113 4616 4617 365 2114 2115 4618 4619 366 2116 2117 4620 4621 367 2118 2119 4622 4623 368 2120 2121 4624 4625 369 2122 2123 4626 4627 370 2124 2125 4628 4629 371 2128 2129 4632 4633 372 2130 2131 4634 4635 373 2134 2135 4638 4639 374 2136 2137 4640 4641 375 2138 2139 4642 4643 376 2140 2141 4644 4645 377 2142 2143 4646 4647 378 2144 2145 4648 4649 379 2146 2147 4650 4651 380 2148 2149 4652 4653 381 2150 2151 4654 4655 382 2152 2153 4656 4657 383 2154 2155 4658 4659 384 2156 2157 4660 4661 385 2158 2159 4662 4663 386 2160 2161 4664 4665 387 2162 2163 4666 4667 388 2164 2165 4668 4669 389 2170 2171 4674 4675 390 2172 2173 4676 4677 391 2174 2175 4678 4679 392 2176 2177 4680 4681 393 2178 2179 4682 4683 394 2180 2181 4684 4685 395 2182 2183 4686 4687 396 2186 2187 4690 4691 397 2190 2191 4694 4695 398 2188 2189 4692 4693 399 2194 2195 4698 4699 400 2192 2193 4696 4697 401 2196 2197 4700 4701 402 2200 2201 4704 4705 403 2198 2199 4702 4703 404 2202 2203 4706 4707 405 2204 2205 4708 4709 406 2206 2207 4710 4711 407 2208 2209 4712 4713 408 2210 2211 4714 4715 409 2212 2213 4716 4717 410 2214 2215 4718 4719 411 2216 2217 4720 4721 412 2218 2219 4722 4723 413 2220 2221 4724 4725 414 2222 2223 4726 4727 415 2224 2225 4728 4729 416 2226 2227 4730 4731 417 2228 2229 4732 4733 418 2230 2231 4734 4735 419 2232 2233 4736 4737 420 2234 2235 4738 4739 421 2236 2237 4740 4741 422 2238 2239 4742 4743 423 2242 2243 4746 4747 424 2244 2245 4748 4749 425 2246 2247 4750 4751 426 2248 2249 4752 4753 427 2250 2251 4754 4755 428 2252 2253 4756 4757 429 2254 2255 4758 4759 430 2256 2257 4760 4761 431 2258 2259 4762 4763 432 2260 2261 4764 4765 433 2262 2263 4766 4767 434 2266 2267 4770 4771 435 2264 2265 4768 4769 436 2268 2269 4772 4773 437 2270 2271 4774 4775 438 2272 2273 4776 4777 439 2274 2275 4778 4779 440 2278 2279 4782 4783 441 2280 2281 4784 4785 442 2282 2283 4786 4787 443 2284 2285 4788 4789 444 2286 2287 4790 4791 445 2288 2289 4792 4793 446 2290 2291 4794 4795 447 2292 2293 4796 4797 448 2294 2295 4798 4799 449 2296 2297 4800 4801 450 2298 2299 4802 4803 451 2304 2305 4808 4809 452 2306 2307 4810 4811 453 2308 2309 4812 4813 454 2310 2311 4814 4815 455 2312 2313 4816 4817 456 2314 2315 4818 4819 457 2316 2317 4820 4821 458 2318 2319 4822 4823 459 2320 2321 4824 4825 460 2322 2323 4826 4827 461 2324 2325 4828 4829 462 2326 2327 4830 4831 463 2328 2329 4832 4833 464 2332 2333 4836 4837 465 2334 2335 4838 4839 466 2338 2339 4842 4843 467 2340 2341 4844 4845 468 2342 2343 4846 4847 469 2344 2345 4848 4849 470 2346 2347 4850 4851 471 2348 2349 4852 4853 472 2350 2351 4854 4855 473 2352 2353 4856 4857 474 2356 2357 4860 4861 475 2354 2355 4858 4859 476 2358 2359 4862 4863 477 2360 2361 4864 4865 478 2362 2363 4866 4867 479 2364 2365 4868 4869 480 2366 2367 4870 4871 481 2368 2369 4872 4873 482 2370 2371 4874 4875 483 2372 2373 4876 4877 484 2374 2375 4878 4879 485 2376 2377 4880 4881 486 2378 2379 4882 4883 487 2380 2381 4884 4885 488 2382 2383 4886 4887 489 2384 2385 4888 4889 490 2386 2387 4890 4891 491 2388 2389 4892 4893 492 2390 2391 4894 4895 493 2392 2393 4896 4897 494 2394 2395 4898 4899 495 2396 2397 4900 4901 496 2398 2399 4902 4903 497 2400 2401 4904 4905 498 2402 2403 4906 4907 499 2404 2405 4908 4909 500 2406 2407 4910 4911 501 2408 2409 4912 4913 502 2410 2411 4914 4915 503 2412 2413 4916 4917 504 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3664 3665 6168 6169 1244 3670 3671 6174 6175 1245 3710 3711 6214 6215 1246 3712 3713 6216 6217 1247 3716 3717 6220 6221 1248 3718 3719 6222 6223 1249 3752 3753 6256 6257 1250 3764 3765 6268 6269 1251 3768 3769 6272 6273 1252 3790 3791 6294 6295 1253 3792 3793 6296 6297 1254 6300 6301 6376 6377 1255 6302 6303 6378 6379 1256 6304 6305 6380 6381 1257 6306 6307 6382 6383 1258 6308 6309 6384 6385 1259 6310 6311 6386 6387 1260 6312 6313 6388 6389 1261 6314 6315 6390 6391 1262 6316 6317 6392 6393 1263 6318 6319 6394 6395 1264 6320 6321 6396 6397 1265 6322 6323 6398 6399 1266 6324 6325 6400 6401 1267 6326 6327 6402 6403 1268 6328 6329 6404 6405 1269 6330 6331 6406 6407 1270 6332 6333 6408 6409 1271 6334 6335 6410 6411 1272 6336 6337 6412 6413 1273 6338 6339 6414 6415 1274 6340 6341 6416 6417 1275 6342 6343 6418 6419 1276 6344 6345 6420 6421 1277 6346 6347 6422 6423 1278 6348 6349 6424 6425 1279 6350 6351 6426 6427 1280 6352 6353 6428 6429 1281 6354 6355 6430 6431 1282 6356 6357 6432 6433 1283 6358 6359 6434 6435 1284 6360 6361 6436 6437 1285 6362 6363 6438 6439 1286 6364 6365 6440 6441 1287 6366 6367 6442 6443 1288 6368 6369 6444 6445 1289 6370 6371 6446 6447 1290 6372 6373 6448 6449 1291 6374 6375 6450 6451

TABLE 5 SEQ ID or. 5′ position 1292 F 1229848 1293 F 1227874 1294 F 1018 1295 F 1229162 1296 F 1588 1297 F 1229711 1298 F 2253 1299 F 369 1300 F 3381 1301 F 1508 1302 F 4042 1303 F 2126 1304 F 5735 1305 F 3843 1306 F 7832 1307 F 5909 1308 F 8887 1309 F 7010 1310 F 10139 1311 F 8175 1312 F 10640 1313 F 8799 1314 F 10997 1315 F 9037 1316 F 12458 1317 F 10572 1318 F 14187 1319 F 12365 1320 F 15529 1321 F 13629 1322 F 17626 1323 F 15699 1324 F 20909 1325 F 19006 1326 F 21800 1327 F 19927 1328 F 23462 1329 F 21557 1330 F 25637 1331 F 23729 1332 F 25997 1333 F 24071 1334 F 26727 1335 F 24828 1336 F 27528 1337 F 25628 1338 F 28643 1339 F 26765 1340 F 29202 1341 F 27313 1342 F 29793 1343 F 27835 1344 F 31488 1345 F 29639 1346 F 31957 1347 F 30050 1348 F 33570 1349 F 31666 1350 F 34564 1351 F 32664 1352 F 35783 1353 F 33875 1354 F 37597 1355 F 35741 1356 F 39135 1357 F 37236 1358 F 38939 1359 F 37038 1360 F 40872 1361 F 38972 1362 F 42825 1363 F 40923 1364 F 43563 1365 F 41652 1366 F 44531 1367 F 42623 1368 F 45150 1369 F 43250 1370 F 45478 1371 F 43579 1372 F 46755 1373 F 44874 1374 F 47347 1375 F 45386 1376 F 47818 1377 F 45897 1378 F 48893 1379 F 46995 1380 F 49907 1381 F 48000 1382 F 51088 1383 F 49169 1384 F 52651 1385 F 50721 1386 F 53065 1387 F 51176 1388 F 53516 1389 F 51611 1390 F 54242 1391 F 52351 1392 F 55058 1393 F 53159 1394 F 56274 1395 F 54348 1396 F 57078 1397 F 55156 1398 F 58343 1399 F 56392 1400 F 61103 1401 F 59177 1402 F 59701 1403 F 57802 1404 F 61887 1405 F 59971 1406 F 62255 1407 F 60348 1408 F 63515 1409 F 61557 1410 F 63657 1411 F 61761 1412 F 64088 1413 F 62196 1414 F 64422 1415 F 62537 1416 F 65072 1417 F 63140 1418 F 65978 1419 F 64088 1420 F 67046 1421 F 65146 1422 F 67466 1423 F 65580 1424 F 68569 1425 F 66686 1426 F 68609 1427 F 66688 1428 F 70423 1429 F 68479 1430 F 71099 1431 F 69206 1432 F 71829 1433 F 69935 1434 F 73745 1435 F 71931 1436 F 76942 1437 F 75022 1438 F 77404 1439 F 75556 1440 F 78133 1441 F 76192 1442 F 79079 1443 F 77122 1444 F 79471 1445 F 77481 1446 F 79670 1447 F 77816 1448 F 80236 1449 F 78356 1450 F 81108 1451 F 79182 1452 F 83024 1453 F 81158 1454 F 83786 1455 F 81886 1456 F 84739 1457 F 82821 1458 F 84866 1459 F 82967 1460 F 85175 1461 F 83240 1462 F 85690 1463 F 83790 1464 F 86397 1465 F 84507 1466 F 88470 1467 F 86563 1468 F 89038 1469 F 87121 1470 F 91017 1471 F 89146 1472 F 93075 1473 F 91147 1474 F 93846 1475 F 91948 1476 F 94410 1477 F 92561 1478 F 95447 1479 F 93541 1480 F 96074 1481 F 94197 1482 F 97706 1483 F 95841 1484 F 98142 1485 F 96292 1486 F 99925 1487 F 98011 1488 F 101229 1489 F 99338 1490 F 101429 1491 F 99552 1492 F 102137 1493 F 100237 1494 F 102600 1495 F 100657 1496 F 103330 1497 F 101429 1498 F 103877 1499 F 101966 1500 F 104336 1501 F 102469 1502 F 108182 1503 F 106280 1504 F 111814 1505 F 109911 1506 F 112412 1507 F 110553 1508 F 113442 1509 F 111571 1510 F 113891 1511 F 112010 1512 F 114990 1513 F 113112 1514 F 115684 1515 F 113776 1516 F 116526 1517 F 114656 1518 F 117731 1519 F 115825 1520 F 118292 1521 F 116389 1522 F 119593 1523 F 117685 1524 F 120231 1525 F 118292 1526 F 122278 1527 F 120382 1528 F 122610 1529 F 120682 1530 F 123309 1531 F 121390 1532 F 126113 1533 F 124213 1534 F 128975 1535 F 127091 1536 F 134603 1537 F 132806 1538 F 136249 1539 F 134352 1540 F 137680 1541 F 135756 1542 F 137680 1543 F 135799 1544 F 138035 1545 F 136135 1546 F 139266 1547 F 137363 1548 F 140208 1549 F 138351 1550 F 141636 1551 F 139735 1552 F 142808 1553 F 140900 1554 F 144272 1555 F 142372 1556 F 145217 1557 F 143335 1558 F 146527 1559 F 144645 1560 F 146965 1561 F 145086 1562 F 147455 1563 F 145501 1564 F 148810 1565 F 146904 1566 F 151964 1567 F 150062 1568 F 154064 1569 F 152113 1570 F 154888 1571 F 152963 1572 F 155418 1573 F 153558 1574 F 156528 1575 F 154606 1576 F 157433 1577 F 155516 1578 F 158771 1579 F 156842 1580 F 159105 1581 F 157219 1582 F 159657 1583 F 157761 1584 F 160240 1585 F 158316 1586 F 160675 1587 F 158778 1588 F 161289 1589 F 159402 1590 F 161918 1591 F 159979 1592 F 162214 1593 F 160297 1594 F 163996 1595 F 162045 1596 F 165189 1597 F 163288 1598 F 166730 1599 F 164828 1600 F 168243 1601 F 166327 1602 F 168907 1603 F 167064 1604 F 169129 1605 F 167294 1606 F 170632 1607 F 168692 1608 F 171229 1609 F 169381 1610 F 171553 1611 F 169614 1612 F 172433 1613 F 170533 1614 F 173217 1615 F 171316 1616 F 174567 1617 F 172680 1618 F 175342 1619 F 173479 1620 F 175709 1621 F 173752 1622 F 176909 1623 F 175009 1624 F 176704 1625 F 174761 1626 F 177608 1627 F 175709 1628 F 179259 1629 F 177384 1630 F 179719 1631 F 177800 1632 F 181629 1633 F 179743 1634 F 182851 1635 F 180952 1636 F 184230 1637 F 182335 1638 F 184870 1639 F 182962 1640 F 185241 1641 F 183348 1642 F 185611 1643 F 183685 1644 F 186336 1645 F 184445 1646 F 188059 1647 F 186171 1648 F 190828 1649 F 188956 1650 F 191294 1651 F 189428 1652 F 192686 1653 F 190788 1654 F 193380 1655 F 191474 1656 F 193388 1657 F 191474 1658 F 193977 1659 F 192059 1660 F 195480 1661 F 193585 1662 F 195868 1663 F 193969 1664 F 197913 1665 F 196013 1666 F 199088 1667 F 197213 1668 F 202776 1669 F 200876 1670 F 204467 1671 F 202497 1672 F 205584 1673 F 203664 1674 F 206940 1675 F 205063 1676 F 207560 1677 F 205587 1678 F 208048 1679 F 206139 1680 F 209923 1681 F 208023 1682 F 210455 1683 F 208569 1684 F 211049 1685 F 209147 1686 F 211596 1687 F 209705 1688 F 212226 1689 F 210311 1690 F 213832 1691 F 211960 1692 F 214866 1693 F 212921 1694 F 215173 1695 F 213307 1696 F 215800 1697 F 213957 1698 F 216489 1699 F 214549 1700 F 216980 1701 F 215100 1702 F 217665 1703 F 215793 1704 F 218039 1705 F 216071 1706 F 218476 1707 F 216560 1708 F 218769 1709 F 216809 1710 F 220020 1711 F 218128 1712 F 221210 1713 F 219275 1714 F 222497 1715 F 220601 1716 F 223292 1717 F 221403 1718 F 223775 1719 F 221877 1720 F 224250 1721 F 222377 1722 F 224906 1723 F 223008 1724 F 225283 1725 F 223418 1726 F 226670 1727 F 224770 1728 F 227849 1729 F 225937 1730 F 228185 1731 F 226269 1732 F 228393 1733 F 226512 1734 F 229334 1735 F 227499 1736 F 230761 1737 F 228846 1738 F 231287 1739 F 229334 1740 F 231731 1741 F 229927 1742 F 232865 1743 F 231027 1744 F 232865 1745 F 231027 1746 F 234315 1747 F 232394 1748 F 234823 1749 F 232865 1750 F 235154 1751 F 233245 1752 F 236429 1753 F 234520 1754 F 237268 1755 F 235271 1756 F 238047 1757 F 236162 1758 F 238636 1759 F 236736 1760 F 239957 1761 F 238047 1762 F 241373 1763 F 239482 1764 F 242017 1765 F 240072 1766 F 242740 1767 F 240829 1768 F 243281 1769 F 241373 1770 F 244244 1771 F 242345 1772 F 246052 1773 F 244179 1774 F 247581 1775 F 245697 1776 F 249216 1777 F 247244 1778 F 251003 1779 F 249137 1780 F 252064 1781 F 250189 1782 F 252900 1783 F 251000 1784 F 253718 1785 F 251855 1786 F 254993 1787 F 253138 1788 F 256414 1789 F 254509 1790 F 257283 1791 F 255383 1792 F 257279 1793 F 255379 1794 F 258061 1795 F 256107 1796 F 259005 1797 F 257128 1798 F 261075 1799 F 259195 1800 F 261551 1801 F 259650 1802 F 262535 1803 F 260611 1804 F 262960 1805 F 261060 1806 F 264509 1807 F 262614 1808 F 265837 1809 F 263925 1810 F 266239 1811 F 264367 1812 F 267185 1813 F 265286 1814 F 267909 1815 F 266037 1816 F 268594 1817 F 266756 1818 F 269299 1819 F 267505 1820 F 271044 1821 F 269121 1822 F 271737 1823 F 269838 1824 F 272558 1825 F 270645 1826 F 273007 1827 F 271098 1828 F 273463 1829 F 271500 1830 F 273922 1831 F 272057 1832 F 275083 1833 F 273094 1834 F 275495 1835 F 273554 1836 F 275739 1837 F 273878 1838 F 276229 1839 F 274371 1840 F 276548 1841 F 274638 1842 F 277098 1843 F 275178 1844 F 277358 1845 F 275448 1846 F 277609 1847 F 275739 1848 F 278314 1849 F 276386 1850 F 279310 1851 F 277385 1852 F 280627 1853 F 278702 1854 F 281471 1855 F 279559 1856 F 282239 1857 F 280288 1858 F 283832 1859 F 281933 1860 F 284384 1861 F 282486 1862 F 285373 1863 F 283473 1864 F 285919 1865 F 284059 1866 F 286742 1867 F 284879 1868 F 287216 1869 F 285329 1870 F 287671 1871 F 285751 1872 F 288273 1873 F 286323 1874 F 288618 1875 F 286685 1876 F 288273 1877 F 286323 1878 F 289723 1879 F 287836 1880 F 289508 1881 F 287667 1882 F 290750 1883 F 288858 1884 F 291142 1885 F 289253 1886 F 291702 1887 F 289812 1888 F 292522 1889 F 290633 1890 F 293035 1891 F 291142 1892 F 293731 1893 F 291786 1894 F 294530 1895 F 292670 1896 F 294367 1897 F 292513 1898 F 296092 1899 F 294209 1900 F 297611 1901 F 295757 1902 F 298027 1903 F 296092 1904 F 298555 1905 F 296582 1906 F 299403 1907 F 297511 1908 F 300409 1909 F 298579 1910 F 301332 1911 F 299433 1912 F 302215 1913 F 300282 1914 F 302492 1915 F 300618 1916 F 303627 1917 F 301730 1918 F 304350 1919 F 302487 1920 F 305173 1921 F 303226 1922 F 306244 1923 F 304350 1924 F 307232 1925 F 305310 1926 F 307799 1927 F 305877 1928 F 309173 1929 F 307301 1930 F 310158 1931 F 308306 1932 F 311020 1933 F 309118 1934 F 311031 1935 F 309126 1936 F 311552 1937 F 309658 1938 F 312510 1939 F 310614 1940 F 313134 1941 F 311255 1942 F 313674 1943 F 311717 1944 F 314490 1945 F 312633 1946 F 315306 1947 F 313355 1948 F 315932 1949 F 314033 1950 F 318434 1951 F 316516 1952 F 320876 1953 F 318949 1954 F 321403 1955 F 319547 1956 F 322084 1957 F 320217 1958 F 322911 1959 F 321049 1960 F 323634 1961 F 321726 1962 F 325117 1963 F 323211 1964 F 326213 1965 F 324254 1966 F 327607 1967 F 325695 1968 F 328162 1969 F 326262 1970 F 328630 1971 F 326723 1972 F 329134 1973 F 327178 1974 F 330734 1975 F 328810 1976 F 332123 1977 F 330252 1978 F 334575 1979 F 332660 1980 F 335884 1981 F 333980 1982 F 337129 1983 F 335202 1984 F 337910 1985 F 335955 1986 F 338746 1987 F 336795 1988 F 339217 1989 F 337362 1990 F 339999 1991 F 338083 1992 F 343144 1993 F 341266 1994 F 343699 1995 F 341813 1996 F 344108 1997 F 342204 1998 F 344851 1999 F 342933 2000 F 346148 2001 F 344219 2002 F 346493 2003 F 344590 2004 F 346815 2005 F 344907 2006 F 347836 2007 F 345956 2008 F 350379 2009 F 348432 2010 F 350856 2011 F 348951 2012 F 352008 2013 F 350106 2014 F 353209 2015 F 351305 2016 F 354224 2017 F 352312 2018 F 354781 2019 F 352871 2020 F 355223 2021 F 353261 2022 F 355393 2023 F 353519 2024 F 358901 2025 F 357001 2026 F 356594 2027 F 354692 2028 F 359240 2029 F 357374 2030 F 359721 2031 F 357763 2032 F 361071 2033 F 359240 2034 F 363605 2035 F 361731 2036 F 364142 2037 F 362246 2038 F 364567 2039 F 362708 2040 F 365039 2041 F 363184 2042 F 365445 2043 F 363517 2044 F 367040 2045 F 365144 2046 F 368825 2047 F 366993 2048 F 369698 2049 F 367760 2050 F 370141 2051 F 368239 2052 F 372329 2053 F 370375 2054 F 372779 2055 F 370881 2056 F 373223 2057 F 371342 2058 F 373939 2059 F 372017 2060 F 374849 2061 F 372953 2062 F 375351 2063 F 373487 2064 F 376316 2065 F 374416 2066 F 377737 2067 F 375828 2068 F 379537 2069 F 377660 2070 F 380033 2071 F 378160 2072 F 380789 2073 F 378889 2074 F 381238 2075 F 379279 2076 F 382969 2077 F 381124 2078 F 383293 2079 F 381425 2080 F 385178 2081 F 383278 2082 F 386271 2083 F 384392 2084 F 386780 2085 F 384891 2086 F 389383 2087 F 387504 2088 F 389901 2089 F 388001 2090 F 390700 2091 F 388732 2092 F 391612 2093 F 389763 2094 F 392346 2095 F 390463 2096 F 392540 2097 F 390639 2098 F 393487 2099 F 391609 2100 F 393904 2101 F 392025 2102 F 394703 2103 F 392782 2104 F 395024 2105 F 393098 2106 F 395705 2107 F 393791 2108 F 397607 2109 F 395705 2110 F 398807 2111 F 396957 2112 F 399848 2113 F 397886 2114 F 400914 2115 F 399008 2116 F 401183 2117 F 399301 2118 F 401964 2119 F 400060 2120 F 403450 2121 F 401527 2122 F 404124 2123 F 402206 2124 F 405765 2125 F 403865 2126 F 407131 2127 F 405243 2128 F 407456 2129 F 405563 2130 F 408841 2131 F 406901 2132 F 410478 2133 F 408573 2134 F 410725 2135 F 408832 2136 F 412263 2137 F 410363 2138 F 414168 2139 F 412268 2140 F 415013 2141 F 413111 2142 F 415636 2143 F 413743 2144 F 417033 2145 F 415114 2146 F 417163 2147 F 415332 2148 F 418186 2149 F 416265 2150 F 420186 2151 F 418259 2152 F 420697 2153 F 418861 2154 F 421313 2155 F 419437 2156 F 422172 2157 F 420342 2158 F 423342 2159 F 421412 2160 F 424008 2161 F 422073 2162 F 424585 2163 F 422711 2164 F 426021 2165 F 424107 2166 F 427407 2167 F 425513 2168 F 427936 2169 F 426053 2170 F 428592 2171 F 426717 2172 F 430475 2173 F 428558 2174 F 431378 2175 F 429417 2176 F 431927 2177 F 430046 2178 F 432609 2179 F 430710 2180 F 433005 2181 F 431082 2182 F 433712 2183 F 431812 2184 F 436521 2185 F 434640 2186 F 436897 2187 F 435057 2188 F 439741 2189 F 437882 2190 F 438296 2191 F 436377 2192 F 440475 2193 F 438538 2194 F 440281 2195 F 438394 2196 F 440989 2197 F 439080 2198 F 442121 2199 F 440252 2200 F 442121 2201 F 440221 2202 F 442780 2203 F 440879 2204 F 443285 2205 F 441384 2206 F 444276 2207 F 442406 2208 F 444472 2209 F 442568 2210 F 444960 2211 F 443040 2212 F 445556 2213 F 443681 2214 F 447565 2215 F 445676 2216 F 448396 2217 F 446496 2218 F 450057 2219 F 448133 2220 F 450444 2221 F 448555 2222 F 450988 2223 F 449054 2224 F 452212 2225 F 450329 2226 F 453450 2227 F 451581 2228 F 454643 2229 F 452718 2230 F 456004 2231 F 454124 2232 F 456785 2233 F 454897 2234 F 457749 2235 F 455856 2236 F 458132 2237 F 456205 2238 F 459216 2239 F 457348 2240 F 460692 2241 F 458792 2242 F 460133 2243 F 458230 2244 F 461228 2245 F 459327 2246 F 462183 2247 F 460269 2248 F 463120 2249 F 461220 2250 F 464355 2251 F 462444 2252 F 464842 2253 F 463010 2254 F 465346 2255 F 463451 2256 F 466061 2257 F 464143 2258 F 466780 2259 F 464842 2260 F 467462 2261 F 465578 2262 F 469419 2263 F 467538 2264 F 471324 2265 F 469419 2266 F 470463 2267 F 468587 2268 F 471822 2269 F 469897 2270 F 472471 2271 F 470610 2272 F 473208 2273 F 471319 2274 F 475143 2275 F 473243 2276 F 477091 2277 F 475181 2278 F 477375 2279 F 475475 2280 F 478473 2281 F 476586 2282 F 479058 2283 F 477158 2284 F 479829 2285 F 477916 2286 F 481237 2287 F 479312 2288 F 481769 2289 F 479903 2290 F 482435 2291 F 480535 2292 F 483976 2293 F 482075 2294 F 484899 2295 F 483029 2296 F 485593 2297 F 483674 2298 F 486401 2299 F 484498 2300 F 486762 2301 F 484859 2302 F 487287 2303 F 485366 2304 F 487487 2305 F 485642 2306 F 488811 2307 F 486942 2308 F 488918 2309 F 487001 2310 F 489740 2311 F 487772 2312 F 490300 2313 F 488400 2314 F 490880 2315 F 488969 2316 F 491167 2317 F 489268 2318 F 492066 2319 F 490096 2320 F 494600 2321 F 492697 2322 F 495778 2323 F 493845 2324 F 496350 2325 F 494396 2326 F 497139 2327 F 495210 2328 F 497504 2329 F 495651 2330 F 498216 2331 F 496381 2332 F 498990 2333 F 497076 2334 F 499284 2335 F 497401 2336 F 499563 2337 F 497644 2338 F 500555 2339 F 498645 2340 F 503868 2341 F 502008 2342 F 504574 2343 F 502741 2344 F 506571 2345 F 504671 2346 F 507498 2347 F 505565 2348 F 507615 2349 F 505777 2350 F 510441 2351 F 508522 2352 F 513523 2353 F 511660 2354 F 516834 2355 F 514938 2356 F 515101 2357 F 513277 2358 F 517031 2359 F 515093 2360 F 517620 2361 F 515698 2362 F 518070 2363 F 516181 2364 F 521162 2365 F 519241 2366 F 523023 2367 F 521123 2368 F 523865 2369 F 522003 2370 F 524373 2371 F 522530 2372 F 526029 2373 F 524115 2374 F 526479 2375 F 524580 2376 F 526756 2377 F 524823 2378 F 528167 2379 F 526263 2380 F 529315 2381 F 527408 2382 F 530372 2383 F 528484 2384 F 531842 2385 F 529945 2386 F 534077 2387 F 532190 2388 F 536335 2389 F 534585 2390 F 536858 2391 F 534931 2392 F 537710 2393 F 535810 2394 F 538105 2395 F 536211 2396 F 538901 2397 F 536979 2398 F 539360 2399 F 537421 2400 F 541059 2401 F 539160 2402 F 542198 2403 F 540335 2404 F 542650 2405 F 540840 2406 F 543589 2407 F 541677 2408 F 546376 2409 F 544486 2410 F 546731 2411 F 544872 2412 F 549480 2413 F 547547 2414 F 550245 2415 F 548328 2416 F 551224 2417 F 549328 2418 F 552433 2419 F 550520 2420 F 554767 2421 F 552882 2422 F 555444 2423 F 553541 2424 F 557979 2425 F 556089 2426 F 557923 2427 F 555988 2428 F 561193 2429 F 559292 2430 F 559671 2431 F 557777 2432 F 561555 2433 F 559655 2434 F 563727 2435 F 561828 2436 F 564714 2437 F 562803 2438 F 566079 2439 F 564180 2440 F 567470 2441 F 565569 2442 F 568454 2443 F 566609 2444 F 569194 2445 F 567291 2446 F 570873 2447 F 568996 2448 F 571678 2449 F 569809 2450 F 571983 2451 F 570083 2452 F 571837 2453 F 569998 2454 F 572927 2455 F 571022 2456 F 574804 2457 F 572868 2458 F 576267 2459 F 574354 2460 F 577925 2461 F 576082 2462 F 578598 2463 F 576721 2464 F 579758 2465 F 577878 2466 F 579620 2467 F 577731 2468 F 579950 2469 F 578022 2470 F 581080 2471 F 579248 2472 F 581459 2473 F 579555 2474 F 582128 2475 F 580221 2476 F 583209 2477 F 581305 2478 F 584650 2479 F 582828 2480 F 585407 2481 F 583467 2482 F 586579 2483 F 584650 2484 F 587655 2485 F 585772 2486 F 587899 2487 F 586058 2488 F 589079 2489 F 587173 2490 F 590446 2491 F 588616 2492 F 592279 2493 F 590407 2494 F 592585 2495 F 590716 2496 F 593527 2497 F 591593 2498 F 594047 2499 F 592210 2500 F 595658 2501 F 593758 2502 F 596225 2503 F 594387 2504 F 596964 2505 F 595006 2506 F 597536 2507 F 595635 2508 F 598383 2509 F 596448 2510 F 599154 2511 F 597254 2512 F 600368 2513 F 598433 2514 F 600665 2515 F 598769 2516 F 602011 2517 F 600087 2518 F 602418 2519 F 600513 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6330 F 593567 6331 F 591675 6332 F 594641 6333 F 592748 6334 F 661934 6335 F 660041 6336 F 706309 6337 F 704409 6338 F 803092 6339 F 801192 6340 F 849060 6341 F 847142 6342 F 913050 6343 F 911152 6344 F 926614 6345 F 924714 6346 F 930121 6347 F 928238 6348 F 986297 6349 F 984362 6350 F 996001 6351 F 994109 6352 F 999731 6353 F 997877 6354 F 1009782 6355 F 1007891 6356 F 1010540 6357 F 1008671 6358 F 1012465 6359 F 1010540 6360 F 1028431 6361 F 1026524 6362 F 1086215 6363 F 1084362 6364 F 1118417 6365 F 1116527 6366 F 1169595 6367 F 1167713 6368 F 1180592 6369 F 1178709 6370 F 1182406 6371 F 1180498 6372 F 1194573 6373 F 1192667 6374 F 1195654 6375 F 1193753 6376 B 26870 6377 B 28721 6378 B 27835 6379 B 29730 6380 B 67456 6381 B 69351 6382 B 70820 6383 B 72708 6384 B 133173 6385 B 135068 6386 B 178637 6387 B 180518 6388 B 208864 6389 B 210727 6390 B 209376 6391 B 211305 6392 B 209483 6393 B 211383 6394 B 210875 6395 B 212766 6396 B 299694 6397 B 301582 6398 B 352312 6399 B 354200 6400 B 420390 6401 B 422291 6402 B 553822 6403 B 555736 6404 B 557050 6405 B 558930 6406 B 594583 6407 B 596527 6408 B 595405 6409 B 597289 6410 B 662614 6411 B 664530 6412 B 707138 6413 B 709063 6414 B 803951 6415 B 805790 6416 B 849771 6417 B 851730 6418 B 913917 6419 B 915796 6420 B 927331 6421 B 929238 6422 B 930857 6423 B 932735 6424 B 986987 6425 B 988912 6426 B 996771 6427 B 998623 6428 B 1000593 6429 B 1002496 6430 B 1010541 6431 B 1012452 6432 B 1011365 6433 B 1013249 6434 B 1013146 6435 B 1015044 6436 B 1029168 6437 B 1031036 6438 B 1087041 6439 B 1088885 6440 B 1119102 6441 B 1121033 6442 B 1170355 6443 B 1172218 6444 B 1181427 6445 B 1183338 6446 B 1183263 6447 B 1185158 6448 B 1195296 6449 B 1197175 6450 B 1196406 6451 B 1198306

TABLE 6 Chromosomal clone Name SEQ ID NO (B) SEQ ID NO (F) region 790313H3# 6452 6648 A 790331B1# 6453 6649 A 790233A9# 6454 6650 A 790031G7# 6455 6651 A 890021E4# 6456 6652 A 790021E11# 6457 6653 A 790332G10# 6458 6654 A 790271B6# 6459 6655 A 790253H6# 6460 6656 A 790214E8# 6461 6657 A 790352D2# 6462 6658 A 790373F2# 6463 6659 A 790424A7# 6464 6660 A 790282F3# 6465 6661 A 790272F5# 6466 6662 A 790424F6# 6467 6663 A 890033H11# 6468 6664 A 790264H10# 6469 6665 A 790293A5# 6470 6666 A 790391E8# 6471 6667 A 890022B8# 6472 6668 A 790332B9# 6473 6669 A 790251B9# 6474 6670 A 790344E8# 6475 6671 B 790323F3# 6476 6672 B 790231G2# 6477 6673 B 790341C5# 6478 6674 B 790332H9# 6479 6675 B 890013A8# 6480 6676 B 790394F2# 6481 6677 B 790222G5# 6482 6678 B 790402A10# 6483 6679 B 790283F6# 6484 6680 B 790041H11# 6485 6681 B 790381C7# 6486 6682 B 790213E1# 6487 6683 B 790211C4# 6488 6684 B 790251B5# 6489 6685 B 790043H9# 6490 6686 B 790303F7# 6491 6687 B 790251G5# 6492 6688 B 790044H7# 6493 6689 B 790022E4# 6494 6690 B 790252A8# 6495 6691 B 790313E9# 6496 6692 B 790264G2# 6497 6693 B 790372A4# 6498 6694 B 790411C2# 6499 6695 B 790322B7# 6500 6696 B 790254F7# 6501 6697 B 790323B12# 6502 6698 B 790263E5# 6503 6699 B 790223C8# 6504 6700 B 790231H2# 6505 6701 B 790324E12# 6506 6702 B 790271D7# 6507 6703 B 790222E8# 6508 6704 B 790083G7# 6509 6705 B 790241D3# 6510 6706 B 790303C8# 6511 6707 B 790283F10# 6512 6708 B 790241B7# 6513 6709 B 790373F10# 6514 6710 B 790362F9# 6515 6711 B 790263H8# 6516 6712 B 790393D10# 6517 6713 B 790313D12# 6518 6714 B 890024C6# 6519 6715 B 890024B10# 6520 6716 B 790212E2# 6521 6717 B 790362E10# 6522 6718 B 790344G11# 6523 6719 B 890011D2# 6524 6720 B 790341B11# 6525 6721 B 790064E10# 6526 6722 B 790212E1# 6527 6723 B 790213G5# 6528 6724 B 790331F2# 6529 6725 B 890024B9# 6530 6726 B 790421F5# 6531 6727 B 890014D11# 6532 6728 B 790373F3# 6533 6729 B 790293D4# 6554 6730 B 790211A3# 6535 6731 B 790211H8# 6556 6732 B 790264E7# 6557 6733 B 790292B11# 6538 6734 B 790312A2# 6539 6735 B 890012D5# 6540 6736 B 790012D12# 6541 6737 B 790291E10# 6542 6738 B 790241C9# 6543 6739 B 790343F1# 6544 6740 B 790241D7# 6545 6741 B 790031H7# 6546 6742 B 790081C4# 6547 6743 B 790013B7# 6548 6744 B 790213F3# 6549 6745 B 790292F9# 6550 6746 B 790423F4# 6551 6747 B 790331F3# 6552 6748 B 790222B10# 6553 6749 B 790261G12# 6554 6750 B 790423G10# 6555 6751 B 790392A9# 6556 6752 B 790331B5# 6557 6753 B 70323H3# 6558 6754 B 890014H8# 6559 6755 B 790231B6# 6560 6756 B 790252F7# 6561 6757 B 790392C10# 6562 6758 B 790021D4# 6563 6759 B 790052D10# 6564 6760 B 790261E3# 6565 6761 B 890023E10# 6566 6762 B 790244B7# 6567 6763 B 790383E1# 6568 6764 B 790401B11# 6569 6765 B 790411B5# 6570 6766 B 790423A11# 6571 6767 B 790031A4# 6572 6768 B 790241G3# 6573 6769 B 790044F7# 6574 6770 B 790252B10# 6575 6771 B 790293F9# 6576 6772 B 790282H3# 6577 6773 B 790381C10# 6578 6774 B 790024H5# 6579 6775 B 790354H7# 6580 6776 B 790411F9# 6581 6777 B 790324G10# 6582 6778 B 790014A5# 6583 6779 B 790381F3# 6584 6780 B 790424D3# 6585 6781 B 790394A10# 6586 6782 B 790423C10# 6587 6783 B 790214D6# 6588 6784 B 790214C4# 6589 6785 B 790014F11# 6590 6786 B 790352F10# 6591 6787 B 790381H6# 6592 6788 B 790282G5# 6593 6789 B 790263C8# 6594 6790 B 890022B4# 6595 6791 B 790283C6# 6596 6792 B 790293B2# 6597 6793 B 790073A3# 6598 6794 B 790313E10# 6599 6795 B 790361D3# 6600 6796 B 790014A11# 6601 6797 B 790254G2# 6602 6798 B 790381C6# 6603 6799 B 790424E3# 6604 6800 B 790421G8# 6605 6801 B 790013C3# 6606 6802 B 790263E8# 6607 6803 B 790373C1# 6608 6804 B 790041C1# 6609 6805 B 790344A7# 6610 6806 B 790271D6# 6611 6807 B 790342H2# 6612 6808 B 890021A6# 6613 6809 B 790381E7# 6614 6810 C 790013G10# 6615 6811 C 790254A4# 6616 6812 C 790213D8# 6617 6813 C 790052A4# 6618 6814 C 790213D3# 6619 6815 C 790394D2# 6620 6816 C 790214D2# 6621 6817 C 790014A4# 6622 6818 C 790324H4# 6623 6819 C 790082B4# 6624 6820 C 790324A6# 6625 6821 C 790424A12# 6626 6822 C 790044G8# 6627 6823 C 790323C6# 6628 6824 C 790312G4# 6629 6825 C 790053C11# 6630 6826 C 890022B7# 6631 6827 C 790392A2# 6632 6828 C 890023D8# 6633 6829 C 790301F1# 6634 6830 C 790343A11# 6635 6831 C 790421A2# 6636 6832 C 790271G2# 6637 6833 C 790302G12# 6638 6834 C 790341E5# 6639 6835 C 790283B6# 6640 6836 C 790222A4# 6641 6837 C 790241B8# 6642 6838 C 790014C2# 6643 6839 C 790402C1# 6644 6840 C 790264E9# 6645 6841 C 790242G4# 6646 6842 C 790422F3# 6647 6843 C

TABLE 7 SEQ ID or. 5′ position 6452 B 29372 6453 B 30198 6454 B 31007 6455 B 31126 6456 B 32735 6457 B 32264 6458 B 32898 6459 B 33582 6460 B 33519 6461 B 34836 6462 B 35795 6463 B 35548 6464 B 35825 6465 B 37239 6466 B 36761 6467 B 37045 6468 B 36761 6469 B 37958 6470 B 38636 6471 B 39813 6472 B 41140 6473 B 40575 6474 B 40526 6475 B 501495 6476 B 502410 6477 B 502586 6478 B 503233 6479 B 503749 6480 B 504488 6481 B 504206 6482 B 504310 6483 B 505455 6484 B 505877 6485 B 506655 6486 B 506513 6487 B 507532 6488 B 507742 6489 B 508050 6490 B 507771 6491 B 509120 6492 B 509646 6493 B 510137 6494 B 510953 6495 B 511165 6496 B 511526 6497 B 511993 6498 B 513012 6499 B 512983 6500 B 512781 6501 B 514155 6502 B 515036 6503 B 515287 6504 B 516292 6505 B 516234 6506 B 516337 6507 B 517347 6508 B 517005 6509 B 516888 6510 B 516234 6511 B 517560 6512 B 517337 6513 B 516756 6514 B 518943 6515 B 519833 6516 B 520123 6517 B 520574 6518 B 520888 6519 B 522154 6520 B 523041 6521 B 522052 6522 B 522217 6523 B 523035 6524 B 524995 6525 B 523567 6526 B 523477 6527 B 523967 6528 B 525211 6529 B 525215 6530 B 526133 6531 B 525674 6532 B 526561 6533 B 526697 6534 B 526715 6535 B 526844 6536 B 527261 6537 B 527503 6538 B 528775 6539 B 528249 6540 B 530307 6541 B 527772 6542 B 529406 6543 B 527752 6544 B 529829 6545 B 529907 6546 B S29574 6547 B 529635 6548 B 530391 6549 B 531516 6550 B 532154 6551 B 532606 6552 B 533407 6553 B 533664 6554 B 533916 6555 B 534707 6556 B 533482 6557 B 534614 6558 B 534935 6559 B 536823 6560 B 535986 6561 B 536143 6562 B 537505 6563 B 537618 6564 B 538165 6565 B 538702 6566 B 540278 6567 B 539156 6568 B 539619 6569 B 540115 6570 B 540724 6571 B 541484 6572 B 540968 6573 B 542062 6574 B 541898 6575 B 543100 6576 B 543846 6577 B 543820 6578 B 544382 6579 B 545158 6580 B 545678 6581 B 545905 6582 B 546683 6583 B 547718 6584 B 547184 6585 B 547684 6586 B 547342 6587 B 548946 6588 B 549071 6589 B 550054 6590 B 549989 6591 B 550426 6592 B 550055 6593 B 550132 6594 B 550132 6595 B 551400 6596 B 551572 6597 B 551468 6598 B 550849 6599 B 552137 6600 B 552325 6601 B 552583 6602 B 553033 6603 B 553629 6604 B 553960 6605 B 553914 6606 B 554354 6607 B 555783 6608 B 555687 6609 B 556441 6610 B 557054 6611 B 556627 6612 B 557292 6613 B 557050 6614 B 815995 6615 B 817104 6616 B 817104 6617 B 816920 6618 B 820464 6619 B 821017 6620 B 821379 6621 B 821504 6622 B 822723 6623 B 823298 6624 B 823380 6625 B 824414 6626 B 824204 6627 B 825288 6628 B 825346 6629 B 825403 6630 B 826237 6631 B 824995 6632 B 826838 6633 B 828146 6634 B 827878 6635 B 827571 6636 B 828472 6637 B 828484 6638 B 828691 6639 B 829507 6640 B 829169 6641 B 828763 6642 B 829769 6643 B 831582 6644 B 830481 6645 B 831468 6646 B 831670 6647 B 832293 6648 F 28484 6649 F 29043 6650 F 29656 6651 F 30157 6652 F 30712 6653 F 31175 6654 F 31658 6655 F 31902 6656 F 32638 6657 F 33203 6658 F 33804 6659 F 34164 6660 F 34426 6661 F 35131 6662 F 35675 6663 F 36097 6664 F 36641 6665 F 36835 6666 F 37236 6667 F 38287 6668 F 38711 6669 F 39117 6670 F 39798 6671 F 500539 6672 F 501016 6673 F 501319 6674 F 501632 6675 F 502155 6676 F 502623 6677 F 503025 6678 F 503681 6679 F 504389 6680 F 504744 6681 F 505468 6682 F 505652 6683 F 505822 6684 F 505833 6685 F 506933 6686 F 507220 6687 F 507559 6688 F 508216 6689 F 508619 6690 F 509329 6691 F 509783 6692 F 510383 6693 F 510729 6694 F 511188 6695 F 511773 6696 F 511869 6697 F 512946 6698 F 513202 6699 F 513821 6700 F 514322 6701 F 514811 6702 F S151O1 6703 F 515611 6704 F 515911 6705 F 516123 6706 F 516169 6707 F S1621S 6708 F 516305 6709 F 517240 6710 F 517993 6711 F 518174 6712 F 518756 6713 F 519133 6714 F 519646 6715 F 520201 6716 F 520563 6717 F 521015 6718 F 521162 6719 F 521543 6720 F 521739 6721 F 522328 6722 F 522567 6723 F 522915 6724 F 523300 6725 F 523791 6726 F 523959 6727 F 524369 6728 F 524801 6729 F 525085 6730 F 525241 6731 F 525738 6732 F 526263 6733 F 526628 6734 F 526779 6735 F 527004 6736 F 527230 6737 F 527381 6738 F 527545 6739 F 527691 6740 F 527932 6741 F 527995 6742 F 528167 6743 F 528610 6744 F 529063 6745 F 529710 6746 F 531140 6747 F 531488 6748 F 531842 6749 F 532064 6750 F 532350 6751 F 532794 6752 F 533117 6753 F 533536 6754 F 533868 6755 F 534200 6756 F 534844 6757 F 535213 6758 F 535678 6759 F 535970 6760 F 536504 6761 F 537013 6762 F 537710 6763 F 538047 6764 F 538353 6765 F 538718 6766 F 539188 6767 F 539471 6768 F 539910 6769 F 540774 6770 F 540962 6771 F 541721 6772 F 542198 6773 F 542644 6774 F 543180 6775 F 543877 6776 F 544601 6777 F 544866 6778 F 545442 6779 F 545948 6780 F 546209 6781 F 546585 6782 F 546960 6783 F 547114 6784 F 547726 6785 F 548045 6786 F 548480 6787 F 548561 6788 F 548775 6789 F 549037 6790 F 549153 6791 F 549597 6792 F 550049 6793 F 550520 6794 F 550890 6795 F 550997 6796 F 551040 6797 F 551247 6796 F 551854 6799 F 552333 6800 F 552603 6801 F 552823 6802 F 553207 6803 F 553898 6804 F 554298 6805 F 554767 6806 F 555323 6807 F 555595 6808 F 555965 6809 F 556248 6810 F 815116 6811 F 815376 6812 F 815849 6813 F 816098 6814 F 818726 6815 F 819337 6816 F 820080 6817 F 820750 6818 F 821170 6819 F 821815 6820 F 822490 6821 F 822789 6822 F 823244 6823 F 823762 6824 F 823964 6825 F 824245 6826 F 824609 6827 F 824948 6828 F 825490 6829 F 826064 6830 F 826405 6831 F 826480 6832 F 827089 6833 F 827418 6834 F 627496 6835 F 827730 6836 F 828180 6837 F 828348 6838 F 828729 6839 F 830099 6840 F 830281 6841 F 830491 6842 F 830550 6843 F 830576

PUBLICATIONS CITED IN THE SPECIFICATION

Adames et al., 1985, Nature, 318:533-538.

Aldous, M. B. et al., 1992, J. Infect. Dis., 166:646-649.

Alexander et al., 1987, Mol. Cell. Biol., 7:1436-1444.

Allan, G. M. et al., 1995, Vet. Microbiol., 44:49-64.

Altschul, S. F. et al., 1990, J. Mol. Biol., 215:403-410.

Altschul et al., 1993, Nature Genetics, 3:266-272.

Altschul et al., 1997, Nucl. Acids Res., 25:3389-3402.

Ansubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY.

Arlinghaus, H. F. et al., 1997, Anal. Biochem., 69, 18, 3747-53.

Bai, M. Et al., 1993, J. Virol., 67:5198-5205.

Barany, F., 1911, PNAS. USA, 88:189-193.

Beattie, K. et al., 1993, Clin. Chem., 39(4):719-721.

Bernoist and Chambon, 1981, Nature, 290:304-310.

Borman, S., 1996, Chem. Eng. News, 74(50):42-43.

Braun, J. et al., 1994 Ann., Rheum Dis 53:100-105.

Brinster et al., 1982, Nature, 296:39-42.

Buckholz, R. G., 1993, Yeast systems for the expression of heterologous gene products. Curr. Op. Biotechnology 4:538-542.

Burg, J. L. et al., 1996, Mol. and Cell. Probes, 10:257-271.

Campbell, L A. et al., 1992 J. Clin. Microbiol. 30:434-439.

Casas-Ciria J. et al., 1996

Chatelier, R. C. et al., 1995, Anal. Biochem., 229(1):112-118.

Chee, M. et al., 1996, Science, 274:610-613.

Chu, B. C. F. et al., 1986, NAR, 14:5591-5603.

Chu, P. W. G. et al., 1993, Virus Research, 27:161-171.

Clark, E. G., 1997, American Association of Swine Practitioners, 499-501.

Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96.

Cote et al., 1983, PNAS USA, 80:2026-2030.

Cserzo, M., Wallin, E., Simon, I. von Heijne G and Elofsson, A., 1997, Prot. Eng., 10:673-676.

DeBoer et al., 1980, Scientific American, 242:74-94.

DeBoer et al., 1983, PNAS USA, 80:21-25.

Derisi, J. et al., 1996, Nature Genet, 14:457-460.

Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Reserach Foundation.

Duck, P. et al., 1990, Biotechniques, 9:142-147.

Dulac, G. C. et al., 1989, Can. J. Vet. Res., 53:431-433.

Edwards, C. P., and Aruffo, A., 1993, Current applications of COS cell based transient expression systems. Curr. Op. Biotechnology 4:558-563.

Edwards, S. et al., 1994, Vet. Rec., 134:680-681.

Erlich, H. A., 1989, In PCR Technology. Principles and Applications for DNA Amplification. New York: Stockton Press.

Falsey, et al., J. Infect. Dis. 168:493-496.

Fanger and Drakeman, 1995, Drug News and Perspectives, 8:133-137.

Felgner, et al., 1987, Proc. Natl. Acad. Sci., 84:7413.

Fodor, S. P. A. et al., 1991, Science, 251:767-771.

Fontes, E. P. B. et al., 1994, J. Biol. Chem., Vol. 269, NO 11:8459-8465.

Fox, G. Et al., 1989, J. Gen. Virol., 70:625-637.

Fraley et al., 1980, J. Biol. Chem., 255:10431.

Gardner et al., 1981, Nucl. Acids Res. 9:2871

Gaydos, C. A. et al., 1994 J. Clin. Microbiol. 32:903-905.

Grayston, J. T. et al., 1986 N. Engl. J. Med., 315:161-168.

Grayston, J T. et al., 1996 Rev., Med Interne 17, 45S-47S.

Gonnet et al., Science, 256:1443-1445.

Grosschedl et al., 1984, Cell, 38:647-658.

Guateli, J. C. et al., 1990, PNAS. USA, 87:1874-1878.

Hackland, A. F. et al., 1994, Arch. Virol., 139:1-22.

Hahn, D L. Et al., 1991 JAMA. 266

Haidl, et al., 1992 N. Engl. J. Med. 326:576-577.

Haidl, et al., Chlamydial infections 1994

Hammer et al., 1987, Science, 235:53-58.

Hanahan, 1985, Nature, 315:115-122.

Hanson, S. F. et al., 1995, Virology, 211:1-9.

Harding, J. C., 1997, American Association of Swine Practitioners, 503.

Harding, R. M. et al., 1993, Journal of General Virology, 74:323-328.

Hashiguchi, K. et al., 1992 J. Laryngol. Otol. 106:208-210.

Hayashi, S. and Wu, H. C., 1992, in N. M. Hooper and A. J. Turner (ed.) Lipid Modification of Proteins: A Practical Approach. Oxford University Press, New York, pp. 261-285.

Heinkoff and Heinkoff, 1993, Proteins, 17:49-61.

Herrera-Estrella et al., 1983, Nature, 303:209-213.

Herrera-Estrella, 1984, Nature, 310:115-120.

Heyraud-Nitschke, F. et al., 1995, Nucleic Acids Research, Vol. 23, NO. 6.

Higgins et al., 1996, Meth. Enzymol., 266:383-402.

Horner, G. W., 1991, Surveillance 18(5):23.

Houbenweyl, 1974, in Meuthode der Organischen Chemie, E.

Wunsch Ed., Volume 15-I et 15-II, Thieme, Stuttgart.

Hsia, R. et al., 1997, Molecular Micorbiology, 25:351-359.

Hueck, C. J., 1998, Molec. Biology Rev., 62:379-433.

Huovinen, P. et al., 1989 Ann., Intern Med 110:612-616.

Huse et al., 1989, Science, 246:1275-1281.

Huygen, K. et al., 1996, Nature Medicine, 2(8):893-898.

Innis, M. A. et al. 1990. in PCR Protocols. A guide to Methods and Applications. San Diego: Academic Press.

Inoue et al., 1987, Nucl. Acids Res., 15:6131-6148.

Inoue et al., 1987, FEBS Lett. 215:327-330.

Jackson, L A. et al., 1997 Am., J. Pathol. 150.:1785-1790.

Jantos et al., 1997, J. Clin. Microbiol., 35(3):620-623.

Kabat E. Et al., 1983, Sequences of Proteins of Immunological Interest, U.S. Dept. Of Health and Human Services.

Kaneda, et al., 1989, Science, 243:375.

Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA, 87:2267-2268

Kelsey et al., 1987, Genes and Devel., 1:161-171.

Kievitis, T. et al., 1991, J. Virol. Methods, 35:273-286.

Kleemola, M. et al., 1988, J. Infect. Dis. 157:230-236.

Kohler, G. et al., 1975, Nature, 256(5517):495-497.

Kollias et al., 1986, Cell, 46:89-94.

Kozbor et al., 1983, Immunol. Today, 4:72.

Krone, J. R. et al., 1997, Anal. Biochem., 244, 1, 124-132.

Krumlauf et al., 1985, Mol. Cell. Biol., 5:1639-1648.

Kuo, C C. et al., 1988, J. Clin. Microbiol. 26:812-815.

Kuo, C C. et al., 1993, J. Infect. Dis. 167:841-849.

Kwoh, D. Y. et al., 1989, PNAS. USA, 86:1173-1177.

Ladany, S. et. al., 1989, J. Clin. Microbiol. 27:2778-2783.

Laitinen, K. et al., 1997. Chlamydia pneumoniae Infection Induces Inflammatory Changes in the Aortas of Rabbits. Infect. Immun. 65:4832-4835.

Lazarowitz, S. G. et al., 1989, The EMBO Journal, Vol. 8 NO. 4:1023-1032.

Leder et al., 1986, Cell, 45:485-495.

Lee, C. A., 1997, Trends Microbiol., 5:148-156.

Leininger, E. et al., 1991, PNAS USA, 88:345-349.

Lipshutz, R. J. et al., 1995, Biotechniques, 19(3):442-447.

Liu, H. et al., 1997, J. Gen. Virol. 78(Pt6):1265-1270.

Livache, T. et al., 1994, NAR, 22(15):2915-2921.

Lockhart, D. J. et al., 1996, Nature Biotechnol., 14:1675-1680.

Longbottom et al., 1998, Infect Immunol., 66:1317-1324.

Luckow, V. A., 1993, Baculovirus systems for the expression of human gene products. Curr. Op. Biotechnology 4:564-572.

Lukacova, M. Et al., 1994, Infect. Immunol. June, 62(6):2270-2276.

MacDonald, 1987, Hepatology, 7:425-515.

Mankertz, A. et al., 1997, J. Virol., 71:2562-2566.

Mason et al., 1986, Science, 234:1372-1378.

Matson, R. S. et al., 1994, Analytical Biochemistry, 217:306-310.

Matthews, J. A. et al., 1988, Anal. Biochem., 169:1-25.

McNeilly, F. et al., 1996, Vet. Immunol. Immunopathol., 49:295-306.

Meehan, B. M. et al., 1997, J. Gen. Virol., 78:221-227.

Mérel, P., 1994, De la PCR aux puces à ADN, Biofutur, 139:58.

Merrifield, R. D., 1966, J. Am. Chem. Soc., 88(21):5051-5052.

Midoux, 1993, Nucleic Acids Research, 21:871-878.

Miele, E. A. et al., 1983, J. Mol. Biol., 171:281-295.

Moazed, T. C. et al., 1997. Murine Model of Chlamydia pneumoniae Infection and Atherosclerosis. J. Infect. Dis. 175:883-890.

Mogram et al., 1985, Nature, 315:338-340.

Mordhorst, C. H. et al., 1992 Eur., J. Clin. Microbiol. Infect Dis 11:617-620.

Morrison et al., 1984, PNAS USA, 81:6851-6855.

Murphy, F. A. et al., 1995, Sixth Report of the International Committee on Taxonomy of Viruses. Springer-Verlag Wien New York.

Nakai, K. and Kanehisa, M., 1991, Proteins, 11:95-110.

Nielsen, H. et al., 1997, Protein Engin., 10:1-6.

Neuberger et al., 1984, Nature, 312:604-608.

ODonell-Maloney, M. J., 1996, Trends Biotechnol., 14:401-407.

Ogawa, H. et al., 1992 J. Laryngol. Oto 106:490-492.

Olins, P. O., and Lee, S. C., 1993, Recent advances in heterologous gene expression in E. coli. Curr. Op. Biotechnology 4:520-525.

Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol., 50:399-409.

Pagano et al., 1967, J. Virol., 1:891.

Pearson and Lipman, 1988, PNAS USA, 85(8):2444-2448.

Peterson, E. M. et al., 1998, Infect. Immunol. Aug., 66(8):3848-3855.

Peterson, E. et al., 1988. Protective Role of Magnesium in the Neutralization by Antibodies of Chlamydia trachomatis Infectivity.

Pierschbacher and Ruoslahti, 1987, J. Biol. Chem., 262:17294-17298.

Pinkert et al., 1987, Genes and Devel., 1:268-276.

Pugsley, A. P., 1993, Microbiol. Rev., 57:50-108.

Puolakkainen, M. et al., 1993 J. Clin. Microbiol. 31:2212-2214.

Readhead et al., 1987, Cell, 48:703-712.

Reeves, P. R. et al., 1996, in Bacterial Polysaccharide Synthesis and Gene Nomenclature, Elsevier Science Ltd., pp. 10071-10078.

Relman, D. et al., 1990, Cell, 61:1375-1382.

Roivainen, M. Et al., 1994, Virology, 203:357-365.

Rolfs, A. et al., 1991, In PCR Topics. Usage of Polymerase Chain reaction in Genetic and Infectious Disease. Berlin: Springer-Verlag.

Salzberg et al., 1998, Nucl. Acids Res., 26:544-548.

Sambrook, J. et al., 1989, In Molecular cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Sanchez-Pescador, R., 1988, J. Clin. Microbiol., 26(10):1934-1938.

Sani, 1985, Nature, 314:283-286.

Sarver et al., 1990, Science, 247:1222-1225.

Schnaitman, C. A. and Klena, J. D., 1993, Microbiol. Rev., 57:655-682.

Schneewind, O. Et al., 1995, Science, 268:103-106.

Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Karlin and Altschul, 1990, PNAS USA, 87:2267-2268.

Segev D., 1992, in <<Non-radioactive Labeling and Detection of Biomolecules>>. Kessler C. Springer Verlag, Berlin, New-York:197-205.

Sheldon, E. L., 1993, Clin. Chem., 39(4):718-719.

Shiver, J. W., 1995, in Vaccines 1995, eds Chanock, R. M. Brown, F. Ginsberg, H. S. & Norrby, E.), pp.95-98, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Shoemaker, D. D. et al., 1996, Nature Genet, 14:450-456.

Shor, A. et. al., 1992 S. Afr. Med. J. 82:158-161.

Sosnowsky et al., 1997, PNAS., 94, 1119-1123.

Struyve, M. et al., 1991, J. Mol. Biol., 218:141-148.

Sundelof, et al., 1993 Scand. J. Infec. Dis. 25:259-261.

Sutcliffe, I. C. and Russell, R. R. B., 1995, J. Bacteriol. 177:1123-1128.

Swift et al., 1984, Cell, 38:639-646.

Takeda et al., 1985, Nature, 314:452-454.

Tascon, R. E et al., 1996, Nature Medicine, 2(8):888-892.

Thom, D. H. et al., 1990 Am. J. Epidemiol 132:248-256.

Thomas, G N. et al., 1997 Scand., J. Infect. Dis. Suppl 104, 30-33.

Tischer, I. et al., 1982, Nature, 295:64-66.

Tischer, I. et al., 1986, Arch. Virol., 91:271-276.

Tischer, I. et al., 1988, Zentralbl Bakteriol Mikrobiol Hyg [A] 270:280-287.

Tischer, I. et al., 1995, Arch. Virol., 140:737-743.

Thompson et al., 1994, Nucl. Acids Res., 22(2):4673-4680.

Urdea, M. S., 1988, Nucleic Acids Research, 11:4937-4957.

Villa-Kamaroff et al., 1978, PNAS USA, 75:3727-3731.

Wagner et al., 1981, PNAS USA, 78:1441-1445.

Walker, G. T. et al., 1992, NAR 20:1691-1696.

Walker, G. T. et al., 1992, PNAS. USA, 89:392-396.

White, B. A. et al., 1997, Methods in Molecular Biology, 67, Humana Press, Towota.

Yamamoto et al., 1980, Cell, 22:787-797.

Yershov, G. et al., 1996, PNAS., USA, 93:4913-4918.

SEQUENCE LISTING The patent contains a lengthy “Sequence Listing” section. A copy of the “Sequence Listing” is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/sequence.html?DocID=06559294B1). An electronic copy of the “Sequence Listing” will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

What is claimed is:
 1. An isolated, recombinant polynucleotide comprising the nucleotide sequence of an open reading frame (ORF) of a Chlamydia pneumoniae genome selected from the group consisting of: a) a nucleotide sequence of ORF195, ORF227, ORF234, ORF1145, ORF1160, ORF1162, ORF1255, ORF1256, ORF1257, ORF1258, or the complement of the nucleotide sequence; and b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:195, 227, 234, 1145, 1160, 1162, 1255, 1256, 1257, 1258, or the complement of the nucleotide sequence.
 2. The isolated, recombinant polynucleotide of claim 1, wherein the nucleotide sequences is selected from the group consisting of the nucleotide sequence of ORF195, ORF227, ORF234, ORF1145, ORF1160, ORF1162, ORF1255, ORF1256, ORF1257, ORF1258, and the complement of the nucleotide sequence.
 3. An isolated, recombinant polynucleotide of claim 1, wherein the nucleotide sequence is selected from the group consisting of the nucleotide sequences encoding the amino acid sequences of SEQ ID NO:195, 227, 234, 1145, 1160, 1162, 1255, 1256, 1257, 1258, and the complement of the nucleotide sequence.
 4. The isolated, recombinant polynucleotide of claim 1, further comprising elements necessary to direct the expression of said nucleotide sequence in a host cell.
 5. A recombinant host cell comprising the polynucleotide of claim 1 or
 4. 6. The polynucleotide of claim 1, 2, or 3, that is found in Chlamydia pneumoniae, but not in Chlamydia trachomatis.
 7. A polynucleotide encoding a fusion protein comprising a polynucleotide sequence according to claim 1, 2, or 3, ligated in frame to a polynucleotide encoding a heterologous polypeptide.
 8. A recombinant vector containing the polynucleotide of claims 1, 2, 3, or
 4. 9. A recombinant vector that contains the polynucleotide of claim
 7. 10. A genetically engineered host cell that contains the polynucleotide of claims 1, 2, 3, or
 4. 11. A genetically engineered host cell that contains the polynucleotide of claim
 7. 12. A method for producing a polypeptide comprising: (a) culturing the genetically engineered host cell of claim 5 under conditions suitable to produce the polypeptide encoded by the polynucleotide; and (b) recovering the polypeptide from the culture.
 13. A method for producing a fusion polypeptide comprising: (a) culturing the genetically engineered host cell of claim 11 under conditions suitable to produce the polypeptide encoded by the polynucleotide; and (b) recovering the polypeptide from the culture. 